Pyrimido-diazepinone compounds and methods of treating disorders

ABSTRACT

The present invention relates to novel pyrimido-diazepinone compounds, methods of modulating protein kinases, including MPS1 (TTK), ERK5 (BMK1, MAPK7), LRKK2, EphA2, polo kinase 1, 2, 3, or 4, Ack1, Ack2, Abl, DCAMKL1, ABL1, Abl mutants, DCAMKL2, ARK5, BRK, MKNK2, FGFR4, TNK1, PLK1, ULK2, PLK4, PRKD1, PRKD2, PRKD3, ROS1, RPS6KA6, TAOK1, TAOK3, TNK2, Bcr-Abl, GAK, cSrc, TPR-Met, Tie2, MET, FGFR3, Aurora, Axl, Bmx, BTK, c-kit, CHK2, Flt3, MST2, p70S6K, PDGFR, PKB, PKC, Raf, ROCK-H, Rsk1, SGK, TrkA, TrkB and TrkC, and the use of such compounds in the treatment of various diseases, disorders or conditions.

RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 14/775,897, filed Sep. 14, 2015, which is a national stageapplication, filed under 35 U.S.C. § 371, of International ApplicationNo. PCT/US2014/030760, filed on Mar. 17, 2014, which claims the benefitof and priority to U.S. Provisional Application No. 61/802,075, filedMar. 15, 2013, the contents of which are incorporated herein byreference in their entireties.

GOVERNMENT SUPPORT

This invention was made with government support under Grant No.GM079575, CA079871, CA114059 awarded by National Institutes of Health.The government has certain rights in the invention

TECHNICAL FIELD

The present invention relates to novel pyrimido-diazepinone compoundswhich are able to modulate protein kinases, including MPS1 (TTK), ERK5(BMK1, MAPK7), polo kinase 1, 2, 3, or 4, Ack1, Ack2, Abl, DCAMKL1,ABL1, Abl mutants, DCAMKL2, ARK5, BRK, MKNK2, FGFR4, TNK1, PLK1, ULK2,PLK4, PRKD1, PRKD2, PRKD3, ROS1, RPS6KA6, TAOK1, TAOK3, TNK2, Bcr-Abl,GAK, cSrc, TPR-Met, Tie2, MET, FGFR3, Aurora, Axl, Bmx, BTK, c-kit,CHK2, Flt3, MST2, p70S6K, PDGFR, PKB, PKC, Raf, ROCK-H, Rsk1, SGK, TrkA,TrkB and TrkC, and the use of such compounds in the treatment of variousdiseases, disorders or conditions.

BACKGROUND OF THE INVENTION

Protein kinases represent a large family of proteins, which play acentral role in the regulation of a wide variety of cellular processesand maintaining control over cellular function. Protein kinasesconstitute a large family of structurally related enzymes that areresponsible for the control of a variety of signal transductionprocesses within the cell (see Hardie, G and Hanks, S. The ProteinKinase Facts Book, I and II, Academic Press, San Diego, Calif.: 1995).Protein kinases are thought to have evolved from a common ancestral genedue to the conservation of their structure and catalytic function.Almost all kinases contain a similar 250-300 amino acid catalyticdomain. The kinases may be categorized into families by the substratesthey phosphorylate (e.g., protein-tyrosine, protein-serine/threonine,lipids etc).

In general, protein kinases mediate intracellular signaling bycatalyzing a phosphoryl transfer from a nucleoside triphosphate to aprotein acceptor that is involved in a signaling pathway. Thesephosphorylation events act as molecular on/off switches that canmodulate or regulate the target protein biological function. Thesephosphorylation events are ultimately triggered in response to a varietyof extracellular and other stimuli. Examples of such stimuli includeenvironmental and chemical stress signals (e.g., shock, heat shock,ultraviolet radiation, bacterial endotoxin, and H2O2), cytokines (e.g.,interleukin-1 (IL-I) and tumor necrosis factor alpha (TNF-a), and growthfactors (e.g., granulocyte macrophage-colony stimulating factor(GM-CSF), and fibroblast growth factor (FGF)). An extracellular stimulusmay affect one or more cellular responses related to cell growth,migration, differentiation, secretion of hormones, activation oftranscription factors, muscle contraction, glucose metabolism, controlof protein synthesis, survival and regulation of the cell cycle.

A partial, non-limiting, list of these kinases include: receptortyrosine kinases such as platelet-derived growth factor receptor kinase(PDGF-R), the nerve growth factor receptor, Trk-A, -B and -C, and thefibroblast growth factor receptor, FGFR3; non-receptor tyrosine kinasessuch Abl and the fusion kinase BCR-Abl, Lck, Csk, Fes, BTK, Bmx andc-src; and serine/threonine kinases such as Aurora, c-RAF, SGK, MAPkinases (e.g., MKK4, MKK6, etc.) and SAPK2α and SAPK2β. Aberrant kinaseactivity has been observed in many disease states including benign andmalignant proliferative disorders as well as diseases resulting frominappropriate activation of the immune and nervous systems.

The novel compounds of this invention inhibit the activity of one ormore protein kinases and are, therefore, expected to be useful in thetreatment of kinase-associated diseases.

SUMMARY OF THE INVENTION

In one aspect, the invention provides a compound of formula I:

or a pharmaceutically acceptable salt, ester or prodrug thereof,

wherein,

A is a single bond or double bond;

R′ is H or alkyl;

L is absent, S, SO, SO₂, or CO;

X is an optional substituent (for example, halogen, —OH, —NO₂, —CN,—NH₂, protected amino, —NH—C₁-C₁₂-alkyl, —NH—C₂-C₁₂-alkenyl,—NH—C₂-C₁₂-alkenyl, —NH—C₃-C₁₂-cycloalkyl, —NH-aryl, —NH-heteroaryl,—NH-heterocycloalkyl, -dialkylamino, -diarylamino, -diheteroarylamino,—O—C₁-C₁₂-alkyl, —O—C₂-C₁₂-alkenyl, —O—C₂-C₁₂-alkenyl,—O—C₃-C₁₂-cycloalkyl, —O-aryl, —O-heteroaryl, —O-heterocycloalkyl,—C(O)—C₁-C₁₂-alkyl, —C(O)—C₂-C₁₂-alkenyl, —C(O)—C₂-C₁₂-alkenyl,—C(O)—C₃-C₁₂-cycloalkyl, —C(O)-aryl, —C(O)-heteroaryl,—C(O)-heterocycloalkyl, —CONH₂, —CONH—C₁-C₁₂-alkyl,—CONH—C₂-C₁₂-alkenyl, —CONH—C₂-C₁₂-alkenyl, —CONH—C₃-C₁₂-cycloalkyl,—CONH-aryl, —CONH-heteroaryl, —CONH-heterocycloalkyl,—OCO₂—C₁-C₁₂-alkyl, —OCO₂—C₂-C₁₂-alkenyl, —OCO₂—C₂-C₁₂-alkenyl,—OCO₂—C₃-C₁₂-cycloalkyl, —OCO₂-aryl, —OCO₂-heteroaryl,—OCO₂-heterocycloalkyl, —OCONH₂, —OCONH—C₁-C₁₂-alkyl,—OCONH—C₂-C₁₂-alkenyl, —OCONH—C₂-C₁₂-alkenyl, —OCONH—C₃-C₁₂-cycloalkyl,—OCONH-aryl, —OCONH-heteroaryl, —OCONH-heterocycloalkyl,—NHC(O)—C₁-C₁₂-alkyl, —NHC(O)—C₂-C₁₂-alkenyl, —NHC(O)—C₂-C₁₂-alkenyl,—NHC(O)—C₃-C₁₂-cycloalkyl, —NHC(O)-aryl, —NHC(O)-heteroaryl,—NHC(O)-heterocycloalkyl, —NHCO₂—C₁-C₁₂-alkyl, —NHCO₂—C₂-C₁₂-alkenyl,—NHCO₂—C₂-C₁₂-alkenyl, —NHCO₂—C₃-C₁₂-cycloalkyl, —NHCO₂-aryl,—NHCO₂-heteroaryl, —NHCO₂-heterocycloalkyl, —NHC(O)NH₂,—NHC(O)NH—C₁-C₁₂-alkyl, —NHC(O)NH—C₂-C₁₂-alkenyl,—NHC(O)NH—C₂-C₁₂-alkenyl, —NHC(O)NH—C₃-C₁₂-cycloalkyl, —NHC(O)NH-aryl,—NHC(O)NH-heteroaryl, —NHC(O)NH-heterocycloalkyl, NHC(S)NH₂,—NHC(S)NH—C₁-C₁₂-alkyl, —NHC(S)NH—C₂-C₁₂-alkenyl,—NHC(S)NH—C₂-C₁₂-alkenyl, —NHC(S)NH—C₃-C₁₂-cycloalkyl, —NHC(S)NH-aryl,—NHC(S)NH-heteroaryl, —NHC(S)NH-heterocycloalkyl, —NHC(NH)NH₂,—NHC(NH)NH—C₁-C₁₂-alkyl, —NHC(NH)NH—C₂-C₁₂-alkenyl,—NHC(NH)NH—C₂-C₁₂-alkenyl, —NHC(NH)NH—C₃-C₁₂-cycloalkyl,—NHC(NH)NH-aryl, —NHC(NH)NH-heteroaryl, —NHC(NH)NH-heterocycloalkyl,—NHC(NH)—C₁-C₁₂-alkyl, —NHC(NH)—C₂-C₁₂-alkenyl, —NHC(NH)—C₂-C₁₂-alkenyl,—NHC(NH)—C₃-C₁₂-cycloalkyl, —NHC(NH)-aryl, —NHC(NH)-heteroaryl,—NHC(NH)-heterocycloalkyl, —C(NH)NH—C₁-C₁₂-alkyl,—C(NH)NH—C₂-C₁₂-alkenyl, —C(NH)NH—C₂-C₁₂-alkenyl,—C(NH)NH—C₃-C₁₂-cycloalkyl, —C(NH)NH-aryl, —C(NH)NH-heteroaryl,—C(NH)NH-heterocycloalkyl, —S(O)—C₁-C₁₂-alkyl, —S(O)—C₂-C₁₂-alkenyl,—S(O)—C₂-C₁₂-alkenyl, —S(O)—C₃-C₁₂-cycloalkyl, —S(O)-aryl,—S(O)-heteroaryl, —S(O)-heterocycloalkyl-SO₂NH₂, —SO₂NH—C₁-C₁₂-alkyl,—SO₂NH—C₂-C₁₂-alkenyl, —SO₂NH—C₂-C₁₂-alkenyl, —SO₂NH—C₃-C₁₂-cycloalkyl,—SO₂NH-aryl, —SO₂NH-heteroaryl, —SO₂NH-heterocycloalkyl,—NHSO₂—C₁-C₁₂-alkyl, —NHSO₂—C₂-C₁₂-alkenyl, —NHSO₂—C₂-C₁₂-alkenyl,—NHSO₂—C₃-C₁₂-cycloalkyl, —NHSO₂-aryl, —NHSO₂-heteroaryl,—NHSO₂-heterocycloalkyl, —CH₂NH₂, —CH₂SO₂CH₃, aryl, arylalkyl,heteroaryl, heteroarylalkyl, heterocycloalkyl, —C₃-C₁₂-cycloalkyl,polyalkoxyalkyl, polyalkoxy, -methoxymethoxy, -methoxyethoxy, —SH,—S—C₁-C₁₂-alkyl, —S—C₂-C₁₂-alkenyl, —S—C₂-C₁₂-alkenyl,—S—C₃-C₁₂-cycloalkyl, —S-aryl, —S-heteroaryl, —S-heterocycloalkyl, ormethylthiomethyl);

R₁ is H, alkyl, alkenyl, alkynyl, each containing 0, 1, 2, or 3heteroatoms selected from O, S, or N; or R₁ is aryl, arylalkyl,heteroaryl, heterocyclic, or carbocyclic; wherein R₁ may be optionallysubstituted;

R₂ is hydrogen, optionally substituted alkyl (including aralkyl),optionally substituted cycloalkyl, and optionally substitutedheterocyclyl; and

R₆ is hydrogen or optionally substituted alkyl.

In another aspect, the invention provides a compound of formula II:

or a pharmaceutically acceptable salt, ester or prodrug thereof,

wherein,

R′ is H or alkyl;

L is absent, S, SO, SO₂, or CO;

X is an optional substituent as defined for formula I;

E is NR₂ or CHR₂;

R₁ is H, alkyl, alkenyl, alkynyl, each containing 0, 1, 2, or 3heteroatoms selected from O, S, or N; or R₁ is aryl, arylalkyl,heteroaryl, heterocyclic, or carbocyclic; wherein R₁ may be optionallysubstituted;

R₂ is, independently for each occurrence, hydrogen, optionallysubstituted alkyl (including aralkyl), optionally substitutedcycloalkyl, and optionally substituted heterocyclyl; and

R₆ is hydrogen or optionally substituted alkyl.

In certain embodiments, E is NR₂. In certain embodiments, R₂ is H or—CH₃.

In another aspect, the invention provides a compound of formula III:

or a pharmaceutically acceptable salt, ester or prodrug thereof,

wherein,

R′ is H or alkyl;

L is absent, S, SO, SO₂, or CO;

X is an optional substituent as defined for formula I;

R₁ is H, alkyl, alkenyl, alkynyl, each containing 0, 1, 2, or 3heteroatoms selected from O, S, or N; or R₁ is aryl, arylalkyl,heteroaryl, heterocyclic, or carbocyclic; wherein R₁ may be optionallysubstituted;

R₂ is, independently for each occurrence, hydrogen, optionallysubstituted alkyl (including aralkyl), optionally substitutedcycloalkyl, and optionally substituted heterocyclyl; and

R₆ is hydrogen or optionally substituted alkyl.

In another aspect, the invention provides a compound of formula IV:

or a pharmaceutically acceptable salt, ester or prodrug thereof,

wherein,

R′ is H or alkyl;

L is absent, S, SO, SO₂, or CO;

R₁ is H, alkyl, alkenyl, alkynyl, each containing 0, 1, 2, or 3heteroatoms selected from O, S, or N; or R₁ is aryl, arylalkyl,heteroaryl, heterocyclic, or carbocyclic; wherein R₁ may be optionallysubstituted;

R₂ is, independently for each occurrence, hydrogen, optionallysubstituted alkyl (including aralkyl), optionally substitutedcycloalkyl, and optionally substituted heterocyclyl; and

R₆ is hydrogen or optionally substituted alkyl.

In another aspect, the invention provides a compound of formula V:

or a pharmaceutically acceptable salt, ester or prodrug thereof,

wherein,

R₂ is hydrogen or optionally substituted alkyl;

R₃ is —OH or —O-(optionally substituted alkyl);

R₄ is hydrogen or optionally substituted alkyl; and

R₆ is hydrogen or optionally substituted alkyl.

In another aspect, the invention provides a compound of Formula VI:

or a pharmaceutically acceptable salt, ester or prodrug thereof,

wherein,

R′ is H or alkyl;

L is absent, S, SO, SO₂, or CO;

X is an optional substituent as defined for formula I;

R₁ is H, alkyl, alkenyl, alkynyl, each containing 0, 1, 2, or 3heteroatoms selected from O, S, or N; or R₁ is aryl, arylalkyl,heteroaryl, heterocyclic, or carbocyclic; wherein R₁ may be optionallysubstituted;

R₂ is, independently for each occurrence, hydrogen, optionallysubstituted alkyl, optionally substituted cycloalkyl, and optionallysubstituted heterocyclyl; or

two X moieties on adjacent atoms of the thiophene ring can form,together with the atoms to which they are attached, a phenyl ring; and

R₆ is hydrogen or optionally substituted alkyl.

In another aspect, the invention provides a compound of Formula VII:

or a pharmaceutically acceptable salt, ester or prodrug thereof,wherein,

R′ is H or alkyl;

L is absent, S, SO, SO₂, or CO;

X is an optional substituent as defined for formula I;

R₁ is H, alkyl, alkenyl, alkynyl, each containing 0, 1, 2, or 3heteroatoms selected from O, S, or N; or R₁ is aryl, arylalkyl,heteroaryl, heterocyclic, or carbocyclic; wherein R₁ may be optionallysubstituted;

R₂ is hydrogen, optionally substituted alkyl, optionally substitutedcycloalkyl, and optionally substituted heterocyclyl; and

R₆ is hydrogen or optionally substituted alkyl.

In another aspect, the invention provides a compound of Formula VIII:

or a pharmaceutically acceptable salt, ester or prodrug thereof,wherein,

R′ is H or alkyl;

L is absent, S, SO, SO₂, or CO;

X is an optional substituent as defined for formula I;

Z is O or S;

R₁ is H, alkyl, alkenyl, alkynyl, each containing 0, 1, 2, or 3heteroatoms selected from O, S, or N; or R₁ is aryl, arylalkyl,heteroaryl, heterocyclic, or carbocyclic; wherein R₁ may be optionallysubstituted;

R₂ is hydrogen, optionally substituted alkyl, optionally substitutedcycloalkyl, and optionally substituted heterocyclyl; and

R₆ is hydrogen or optionally substituted alkyl.

In another aspect, the invention provides a compound of Formula IX:

or a pharmaceutically acceptable salt, ester or prodrug thereof,wherein,

A is a single bond or double bond;

R′ is H or alkyl;

L is absent, S, SO, SO₂, or CO;

Y is hydrogen, optionally substituted alkyl, optionally substitutedcycloalkyl, and optionally substituted heterocyclyl;

R₁ is H, alkyl, alkenyl, alkynyl, each containing 0, 1, 2, or 3heteroatoms selected from O, S, or N; or R₁ is aryl, arylalkyl,heteroaryl, heterocyclic, or carbocyclic; wherein R₁ may be optionallysubstituted;

R₂ and R₂′ are each independently hydrogen, optionally substitutedalkyl, optionally substituted cycloalkyl, and optionally substitutedheterocyclyl; or Y and R₂′ can form, together with the atoms to whichthey are attached, a five-membered ring; and

R₆ is hydrogen or optionally substituted alkyl.

In another aspect, the invention provides a method of treating a diseasein a subject comprising administering to the subject a compound,pharmaceutically acceptable salt, ester or prodrug of formulae I-IX (orformulae A or F).

In another aspect, the invention provides a method of treating akinase-mediated disorder in a subject comprising: administering to thesubject identified as in need thereof a compound, pharmaceuticallyacceptable salt, ester or prodrug of formulae I-IX (or formulae A or F).

In another aspect, the invention provides a method for reducingkinase-dependent cell growth comprising contacting a cell with a kinaseinhibitor compound of formulae I-IX (or formulae A or F).

In other aspects, the invention provides a method of inhibiting a kinasein a cell or in a subject identified as in need of such treatment,comprising administering a compound of formulae I-IX (or formulae A orF).

In another aspect, the invention provides a kit comprising a compoundcapable of inhibiting kinase activity selected from one or morecompounds of formulae I-IX (or formulae A or F), and instructions foruse in treating cancer.

In another aspect, the invention provides a pharmaceutical compositioncomprising a compound of formulae I-IX (or formulae A or F), or apharmaceutically acceptable ester, salt, or prodrug thereof, togetherwith a pharmaceutically acceptable carrier.

In one aspect, the invention provides a method of synthesizing acompound of formulae I-IX (or formulae A or F).

DESCRIPTION OF THE DRAWINGS

FIG. 1. Summary of SAR for benzo[e]pyrimido-[5,4-b]diazepine-6(11H)-onesas ERK5 inhibitors and LRRK2 inhibitors.

FIG. 2A-FIG. 2B. Compound 24 inhibits LRRK2 in cells, but 26 does not.In FIG. 2A, HEK293 cells stably expressing wild-type GFP-LRRK2,GFP-LRRK2[G2019S], GFP-LRRK2[G2019S+A2016T], and GFP-LRRK2[A2016T] weretreated with DMSO or increasing concentrations of compound 24 for 90min. Cell lysates were subjected to immunoblotting for detection ofLRRK2 phosphorylated at Ser910 and Ser935 and for total LRRK2. FIG. 2Bshows similar results as in FIG. 2A except 26 was used at the indicatedconcentration.

FIG. 3A-FIG. 3B. Compound 24 effectively inhibits endogenously expressedLRRK2, but compound 26 does not. Endogenous LRRK2 from EBV immortalizedhuman lymphoblastoid cells from a control subject and a Parkinson'sdisease patient homozygous for the LRRK2[G2019S] mutation. Aftertreatment of the cells with DMSO or the indicated concentration ofcompound 24 (or 26) for 90 min, cell lysates were subjected toimmunoblot analysis with the purified indicated antibody for westernanalysis. Immunoblots were performed in duplicate, and results wererepresentative of at least two independent experiments.

FIG. 4A, FIG. 4B, and FIG. 4C. Docking model of 26 bound to LRRK2 fromthree different viewing angles. The N-terminal lobe of the LRRK2 modelis shown in pink, and the C-terminal lobe in green. 26 is shown inyellow.

DETAILED DESCRIPTION OF THE INVENTION Definitions

Listed below are definitions of various terms used to describe thisinvention. These definitions apply to the terms as they are usedthroughout this specification and claims, unless otherwise limited inspecific instances, either individually or as part of a larger group.

The term “alkyl,” as used herein, refers to saturated, straight- orbranched-chain hydrocarbon radicals containing, in certain embodiments,between one and six, or one and eight carbon atoms, respectively.Examples of C₁-C₆ alkyl radicals include, but are not limited to,methyl, ethyl, propyl, isopropyl, n-butyl, tert-butyl, neopentyl,n-hexyl radicals; and examples of C₁-C₈ alkyl radicals include, but arenot limited to, methyl, ethyl, propyl, isopropyl, n-butyl, tert-butyl,neopentyl, n-hexyl, heptyl, octyl radicals.

The term “alkenyl,” as used herein, denotes a monovalent group derivedfrom a hydrocarbon moiety containing, in certain embodiments, from twoto six, or two to eight carbon atoms having at least one carbon-carbondouble bond. The double bond may or may not be the point of attachmentto another group. Alkenyl groups include, but are not limited to, forexample, ethenyl, propenyl, butenyl, 1-methyl-2-buten-1-yl, heptenyl,octenyl and the like.

The term “alkynyl,” as used herein, denotes a monovalent group derivedfrom a hydrocarbon moiety containing, in certain embodiments, from twoto six, or two to eight carbon atoms having at least one carbon-carbontriple bond. The alkynyl group may or may not be the point of attachmentto another group. Representative alkynyl groups include, but are notlimited to, for example, ethynyl, 1-propynyl, 1-butynyl, heptynyl,octynyl and the like.

The term “alkoxy” refers to an —O-alkyl radical.

The term “aryl,” as used herein, refers to a mono- or poly-cycliccarbocyclic ring system having one or more aromatic rings, fused ornon-fused, including, but not limited to, phenyl, naphthyl,tetrahydronaphthyl, indanyl, idenyl and the like.

The term “aralkyl,” as used herein, refers to an alkyl residue attachedto an aryl ring. Examples include, but are not limited to, benzyl,phenethyl and the like.

The term “cycloalkyl,” as used herein, denotes a monovalent groupderived from a monocyclic or polycyclic saturated or partially unsaturedcarbocyclic ring compound. Examples of C₃-C₈-cycloalkyl include, but notlimited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,cyclopentyl and cyclooctyl; and examples of C₃-C₁₂-cycloalkyl include,but not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,bicyclo [2.2.1] heptyl, and bicyclo [2.2.2] octyl. Also contemplated area monovalent group derived from a monocyclic or polycyclic carbocyclicring compound having at least one carbon-carbon double bond by theremoval of a single hydrogen atom. Examples of such groups include, butare not limited to, cyclopropenyl, cyclobutenyl, cyclopentenyl,cyclohexenyl, cycloheptenyl, cyclooctenyl, and the like.

The term “heteroaryl,” as used herein, refers to a mono- or poly-cyclic(e.g., bi-, or tri-cyclic or more) fused or non-fused, radical or ringsystem having at least one aromatic ring, having from five to ten ringatoms of which one ring atoms is selected from S, O and N; zero, one ortwo ring atoms are additional heteroatoms independently selected from S,O and N; and the remaining ring atoms are carbon. Heteroaryl includes,but is not limited to, pyridinyl, pyrazinyl, pyrimidinyl, pyrrolyl,pyrazolyl, imidazolyl, thiazolyl, oxazolyl, isooxazolyl, thiadiazolyl,oxadiazolyl, thiophenyl, furanyl, quinolinyl, isoquinolinyl,benzimidazolyl, benzooxazolyl, quinoxalinyl, and the like.

The term “heteroaralkyl,” as used herein, refers to an alkyl residueresidue attached to a heteroaryl ring. Examples include, but are notlimited to, pyridinylmethyl, pyrimidinylethyl and the like.

The term “heterocycloalkyl,” as used herein, refers to a non-aromatic3-, 4-, 5-, 6- or 7-membered ring or a bi- or tri-cyclic group fused ofnon-fused system, where (i) each ring contains between one and threeheteroatoms independently selected from oxygen, sulfur and nitrogen,(ii) each 5-membered ring has 0 to 1 double bonds and each 6-memberedring has 0 to 2 double bonds, (iii) the nitrogen and sulfur heteroatomsmay optionally be oxidized, (iv) the nitrogen heteroatom may optionallybe quaternized, and (iv) any of the above rings may be fused to abenzene ring. Representative heterocycloalkyl groups include, but arenot limited to, [1,3]dioxolane, pyrrolidinyl, pyrazolinyl,pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl,oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl,isothiazolidinyl, and tetrahydrofuryl.

The term “alkylamino” refers to a group having the structure —NH(C₁-C₁₂alkyl) where C₁-C₁₂ alkyl is as previously defined.

The term “acyl” includes residues derived from acids, including but notlimited to carboxylic acids, carbamic acids, carbonic acids, sulfonicacids, and phosphorous acids. Examples include aliphatic carbonyls,aromatic carbonyls, aliphatic sulfonyls, aromatic sulfinyls, aliphaticsulfinyls, aromatic phosphates and aliphatic phosphates. Examples ofaliphatic carbonyls include, but are not limited to, acetyl, propionyl,2-fluoroacetyl, butyryl, 2-hydroxy acetyl, and the like.

In accordance with the invention, any of the aryls, substituted aryls,heteroaryls and substituted heteroaryls described herein, can be anyaromatic group. Aromatic groups can be substituted or unsubstituted.

The terms “halo” and “halogen,” as used herein, refer to an atomselected from fluorine, chlorine, bromine and iodine.

As described herein, compounds of the invention may optionally besubstituted with one or more substituents, such as are illustratedgenerally above, or as exemplified by particular classes, subclasses,and species of the invention. It will be appreciated that the phrase“optionally substituted” is used interchangeably with the phrase“substituted or unsubstituted.” In general, the term “substituted”,whether preceded by the term “optionally” or not, refers to thereplacement of hydrogen radicals in a given structure with the radicalof a specified substituent. Unless otherwise indicated, an optionallysubstituted group may have a substituent at each substitutable positionof the group, and when more than one position in any given structure maybe substituted with more than one substituent selected from a specifiedgroup, the substituent may be either the same or different at everyposition. The terms “optionally substituted”, “optionally substitutedalkyl,” “optionally substituted “optionally substituted alkenyl,”“optionally substituted alkynyl”, “optionally substituted cycloalkyl,”“optionally substituted cycloalkenyl,” “optionally substituted aryl”,“optionally substituted heteroaryl,” “optionally substituted aralkyl”,“optionally substituted heteroaralkyl,” “optionally substitutedheterocycloalkyl,” and any other optionally substituted group as usedherein, refer to groups that are substituted or unsubstituted byindependent replacement of one, two, or three or more of the hydrogenatoms thereon with substituents including, but not limited to:

—F, —Cl, —Br, —I,

—OH, protected hydroxy,

—NO₂, —CN,

—NH₂, protected amino, —NH—C₁-C₁₂-alkyl, —NH—C₂-C₁₂-alkenyl,—NH—C₂-C₁₂-alkenyl, —NH—C₃-C₁₂-cycloalkyl, —NH-aryl, —NH-heteroaryl,—NH-heterocycloalkyl, -dialkylamino, -diarylamino, -diheteroarylamino,

—O—C₁-C₁₂-alkyl, —O—C₂-C₁₂-alkenyl, —O—C₂-C₁₂-alkenyl,—O—C₃-C₁₂-cycloalkyl, —O-aryl, —O-heteroaryl, —O-heterocycloalkyl,

—C(O)—C₁-C₁₂-alkyl, —C(O)—C₂-C₁₂-alkenyl, —C(O)—C₂-C₁₂-alkenyl,—C(O)—C₃-C₁₂-cycloalkyl, —C(O)-aryl, —C(O)-heteroaryl,—C(O)-heterocycloalkyl, —CONH₂, —CONH—C₁-C₁₂-alkyl,—CONH—C₂-C₁₂-alkenyl, —CONH—C₂-C₁₂-alkenyl, —CONH—C₃-C₁₂-cycloalkyl,—CONH-aryl, —CONH-heteroaryl, —CONH-heterocycloalkyl,

—OCO₂—C₁-C₁₂-alkyl, —OCO₂—C₂-C₁₂-alkenyl, —OCO₂—C₂-C₁₂-alkenyl,—OCO₂—C₃-C₁₂-cycloalkyl, —OCO₂-aryl, —OCO₂-heteroaryl,—OCO₂-heterocycloalkyl, —OCONH₂, —OCONH—C₁-C₁₂-alkyl,—OCONH—C₂-C₁₂-alkenyl, —OCONH—C₂-C₁₂-alkenyl, —OCONH—C₃-C₁₂-cycloalkyl,—OCONH-aryl, —OCONH-heteroaryl, —OCONH-heterocycloalkyl,—NHC(O)—C₁-C₁₂-alkyl, —NHC(O)—C₂-C₁₂-alkenyl, —NHC(O)—C₂-C₁₂-alkenyl,—NHC(O)—C₃-C₁₂-cycloalkyl, —NHC(O)-aryl, —NHC(O)-heteroaryl,—NHC(O)-heterocycloalkyl, —NHCO₂—C₁-C₁₂-alkyl, —NHCO₂—C₂-C₁₂-alkenyl,—NHCO₂—C₂-C₁₂-alkenyl, —NHCO₂—C₃-C₁₂-cycloalkyl, —NHCO₂-aryl,—NHCO₂-heteroaryl, —NHCO₂-heterocycloalkyl, —NHC(O)NH₂,—NHC(O)NH—C₁-C₁₂-alkyl, —NHC(O)NH—C₂-C₁₂-alkenyl,—NHC(O)NH—C₂-C₁₂-alkenyl, —NHC(O)NH—C₃-C₁₂-cycloalkyl, —NHC(O)NH-aryl,—NHC(O)NH-heteroaryl, —NHC(O)NH-heterocycloalkyl, NHC(S)NH₂,—NHC(S)NH—C₁-C₁₂-alkyl, —NHC(S)NH—C₂-C₁₂-alkenyl,—NHC(S)NH—C₂-C₁₂-alkenyl, —NHC(S)NH—C₃-C₁₂-cycloalkyl, —NHC(S)NH-aryl,—NHC(S)NH-heteroaryl, —NHC(S)NH-heterocycloalkyl, —NHC(NH)NH₂,—NHC(NH)NH—C₁-C₁₂-alkyl, —NHC(NH)NH—C₂-C₁₂-alkenyl,—NHC(NH)NH—C₂-C₁₂-alkenyl, —NHC(NH)NH—C₃-C₁₂-cycloalkyl,—NHC(NH)NH-aryl, —NHC(NH)NH-heteroaryl, —NHC(NH)NH-heterocycloalkyl,—NHC(NH)—C₁-C₁₂-alkyl, —NHC(NH)—C₂-C₁₂-alkenyl, —NHC(NH)—C₂-C₁₂-alkenyl,—NHC(NH)—C₃-C₁₂-cycloalkyl, —NHC(NH)-aryl, —NHC(NH)-heteroaryl,—NHC(NH)-heterocycloalkyl,

—C(NH)NH—C₁-C₁₂-alkyl, —C(NH)NH—C₂-C₁₂-alkenyl, —C(NH)NH—C₂-C₁₂-alkenyl,—C(NH)NH—C₃-C₁₂-cycloalkyl, —C(NH)NH-aryl, —C(NH)NH-heteroaryl,—C(NH)NH-heterocycloalkyl,

—S(O)—C₁-C₁₂-alkyl, —S(O)—C₂-C₁₂-alkenyl, —S(O)—C₂-C₁₂-alkenyl,—S(O)—C₃-C₁₂-cycloalkyl, —S(O)-aryl, —S(O)-heteroaryl,—S(O)-heterocycloalkyl-SO₂NH₂, —SO₂NH—C₁-C₁₂-alkyl,—SO₂NH—C₂-C₁₂-alkenyl, —SO₂NH—C₂-C₁₂-alkenyl, —SO₂NH—C₃-C₁₂-cycloalkyl,—SO₂NH-aryl, —SO₂NH-heteroaryl, —SO₂NH-heterocycloalkyl,

—NHSO₂—C₁-C₁₂-alkyl, —NHSO₂—C₂-C₁₂-alkenyl, —NHSO₂—C₂-C₁₂-alkenyl,—NHSO₂—C₃-C₁₂-cycloalkyl, —NHSO₂-aryl, —NHSO₂-heteroaryl,—NHSO₂-heterocycloalkyl,

—CH₂NH₂, —CH₂SO₂CH₃, -aryl, -arylalkyl, -heteroaryl, -heteroarylalkyl,-heterocycloalkyl, —C₃-C₁₂-cycloalkyl, polyalkoxyalkyl, polyalkoxy,-methoxymethoxy, -methoxyethoxy, —SH, —S—C₁-C₁₂-alkyl,—S—C₂-C₁₂-alkenyl, —S—C₂-C₁₂-alkenyl, —S—C₃-C₁₂-cycloalkyl, —S-aryl,—S-heteroaryl, —S-heterocycloalkyl, or methylthiomethyl.

It is understood that the aryls, heteroaryls, alkyls, and the like canbe further substituted.

The term “cancer” includes, but is not limited to, the followingcancers: epidermoid Oral: buccal cavity, lip, tongue, mouth, pharynx;Cardiac: sarcoma (angiosarcoma, fibrosarcoma, rhabdomyosarcoma,liposarcoma), myxoma, rhabdomyoma, fibroma, lipoma and teratoma; Lung:bronchogenic carcinoma (squamous cell or epidermoid, undifferentiatedsmall cell, undifferentiated large cell, adenocarcinoma), alveolar(bronchiolar) carcinoma, bronchial adenoma, sarcoma, lymphoma,chondromatous hamartoma, mesothelioma; Gastrointestinal: esophagus(squamous cell carcinoma, larynx, adenocarcinoma, leiomyosarcoma,lymphoma), stomach (carcinoma, lymphoma, leiomyosarcoma), pancreas(ductal adenocarcinoma, insulinoma, glucagonoma, gastrinoma, carcinoidtumors, vipoma), small bowel or small intestines (adenocarcinoma,lymphoma, carcinoid tumors, Karposi's sarcoma, leiomyoma, hemangioma,lipoma, neurofibroma, fibroma), large bowel or large intestines(adenocarcinoma, tubular adenoma, villous adenoma, hamartoma,leiomyoma), colon, colon-rectum, colorectal; rectum, Genitourinarytract: kidney (adenocarcinoma, WiIm's tumor [nephroblastoma], lymphoma,leukemia), bladder and urethra (squamous cell carcinoma, transitionalcell carcinoma, adenocarcinoma), prostate (adenocarcinoma, sarcoma),testis (seminoma, teratoma, embryonal carcinoma, teratocarcinoma,choriocarcinoma, sarcoma, interstitial cell carcinoma, fibroma,fibroadenoma, adenomatoid tumors, lipoma); Liver: hepatoma(hepatocellular carcinoma), cholangiocarcinoma, hepatoblastoma,angiosarcoma, hepatocellular adenoma, hemangioma, biliary passages;Bone: osteogenic sarcoma (osteosarcoma), fibrosarcoma, malignant fibroushistiocytoma, chondrosarcoma, Ewing's sarcoma, malignant lymphoma(reticulum cell sarcoma), multiple myeloma, malignant giant cell tumorchordoma, osteochronfroma (osteocartilaginous exostoses), benignchondroma, chondroblastoma, chondromyxofibroma, osteoid osteoma andgiant cell tumors; Nervous system: skull (osteoma, hemangioma,granuloma, xanthoma, osteitis deformans), meninges (meningioma,meningiosarcoma, gliomatosis), brain (astrocytoma, medulloblastoma,glioma, ependymoma, germinoma [pinealoma], glioblastoma multiform,oligodendroglioma, schwannoma, retinoblastoma, congenital tumors),spinal cord neurofibroma, meningioma, glioma, sarcoma); Gynecological:uterus (endometrial carcinoma), cervix (cervical carcinoma, pre-tumorcervical dysplasia), ovaries (ovarian carcinoma [serouscystadenocarcinoma, mucinous cystadenocarcinoma, unclassifiedcarcinoma], granulosa-thecal cell tumors, Sertoli-Leydig cell tumors,dysgerminoma, malignant teratoma), vulva (squamous cell carcinoma,intraepithelial carcinoma, adenocarcinoma, fibrosarcoma, melanoma),vagina (clear cell carcinoma, squamous cell carcinoma, botryoid sarcoma(embryonal rhabdomyosarcoma), fallopian tubes (carcinoma), breast;Hematologic: blood (myeloid leukemia [acute and chronic], acutelymphoblastic leukemia, chronic lymphocytic leukemia, myeloproliferativediseases, multiple myeloma, myelodysplastic syndrome), Hodgkin'sdisease, non-Hodgkin's lymphoma [malignant lymphoma] hairy cell;lymphoid disorders; Skin: malignant melanoma, basal cell carcinoma,squamous cell carcinoma, Karposi's sarcoma, keratoacanthoma, molesdysplastic nevi, lipoma, angioma, dermatofibroma, keloids, psoriasis,Thyroid gland: papillary thyroid carcinoma, follicular thyroidcarcinoma; medullary thyroid carcinoma, undifferentiated thyroid cancer,multiple endocrine neoplasia type 2A, multiple endocrine neoplasia type2B, familial medullary thyroid cancer, pheochromocytoma, paraganglioma;and Adrenal glands: neuroblastoma. Thus, the term “cancerous cell” asprovided herein, includes a cell afflicted by any one of theabove-identified conditions.

The term “Kinase Panel” is a list of kinases comprising MPS1 (TTK), ERK5(BMK1, MAPK7), polo kinase 1, 2, 3, or 4, Ack1, Ack2, Abl, DCAMKL1,ABL1, Abl mutants, DCAMKL2, ARK5, BRK, MKNK2, FGFR4, TNK1, PLK1, ULK2,PLK4, PRKD1, PRKD2, PRKD3, ROS1, RPS6KA6, TAOK1, TAOK3, TNK2, Bcr-Abl,GAK, cSrc, TPR-Met, Tie2, MET, FGFR3, Aurora, Axl, Bmx, BTK, c-kit,CHK2, Flt3, MST2, p70S6K, PDGFR, PKB, PKC, Raf, ROCK-H, Rsk1, SGK, TrkA,TrkB, TrkC, AAK1, ABL1, ABL1(E255K), ABL1(F317I), ABL1(F317L),ABL1(H396P), ABL1(M351T), ABL1(Q252H), ABL1(T3151), ABL1(Y253F), ABL2,ACVR1, ACVR1B, ACVR2A, ACVR2B, ACVRL1, ADCK3, ADCK4, AKT1, AKT2, AKT3,ALK, AMPK-alpha1, AMPK-alpha2, ANKK1, ARK5, ASK1, ASK2, AURKA, AURKB,AURKC, AXL, BIKE, BLK, BMPR1A, BMPR1B, BMPR2, BMX, BRAF, BRAF(V600E),BRK, BRSK1, BRSK2, BTK, CAMK1, CAMK1D, CAMK1G, CAMK2A, CAMK2D, CAMK2G,CAMK4, CAMKK1, CAMKK2, CDC2L1, CDC2L2, CDK11, CDK2, CDK3, CDK5, CDK7,CDK8, CDK9, CDKL2, CDKL3, CDKL5, CHECK1, CHEK2, CIT, CLK1, CLK2, CLK3,CLK4, CSF1R, CSK, CSNK1A1L, CSNK1D, CSNK1E, CSNK1G1, CSNK1G3, CSNK2A1,CSNK2A2, CTK, DAPK1, DAPK2, DAPK3, DCAMKL1, DCAMKL2, DCAMKL3, DDR1,DDR2, DLK, DMPK, DMPK2, DRAK1, DRAK2, DYRK1A, DYRK1B, DYRK2, EGFR, EGFR(E746-A750DEL), EGFR (G719C), EGFR (G719S), EGFR(L747-E749del, A750P),EGFR(L747-S752del, P753S), EGFR(L747-T751del,Sins), EGFR(L858R),EGFR(L858R,T790M), EGFR(L861Q), EGFR(5752-1759del), EPHA1, EPHA2, EPHA3,EPHA4, EPHA5, EPHA6, EPHA7, EPHA8, EPHB1, EPHB2, EPHB3, EPHB4, EPHB6,ERBB2, ERBB3, ERBB4, ERK1, ERK2, ERK5, ERK4, ERK5, ERK5, ERN1, FAK, FER,FES, FGFR1, FGFR2, FGFR3, FGFR3(G697C), FGFR4, FGR, FLT1, FLT3,FLT3(D835H), FLT3(D835Y), FLT3(ITD), FLT3(K663Q), FLT3(N841I), FLT4,FRK, FYN, GAK, GCN2(Kin.Dom.2,S808G), GRK1, GRK4, GRK7, GSK3A, GSK3B,HCK, HIPK1, HIPK2, HIPK3, HIPK4, HPK1, HUNK, ICK, IGF1R, IKK-ALPHA,IKK-BETA, IKK-EPSILON, INSR, INSRR, IRAK1, IRAK3, ITK,JAK1(JH1domain-catalytic), JAK1(JH2domain-pseudokinase),JAK2(JH1domain-catalytic), JAK3(JH1domain-catalytic), JNK1, JNK2, JNK3,KIT, KIT(D816V), KIT(L576P), KIT(V559D), KIT(V559D,T670I),KIT(V559D,V654A), LATS1, LATS2, LCK, LIMK1, LIMK2, LKB1, LOK, LTK, LYN,LZK, MAK, MAP3K1, MAP2K15, MAP3K2, MAP3K3, MAP3K4, MAP4K2, MAP4K3,MAP4K5, MAPKAPK2, MAPKAPK5, MARK1, MARK2, MARK3, MARK4, MAST1, MEK1,MEK2, MEK3, MEK4, MEK6, MELK, MERTK, MET, MET(M1250T), MET(Y1235D),MINK, MKNK1, MKNK2, MLCK, MLK1, MLK2, MLK3, MRCKA, MRCKB, MST1, MST1R,MST2, MST3, MST4, MUSK, MYLK, MYLK2, MYO3A, MYO3B, NDR1, NDR2, NEK1,NEK2, NEK5, NEK6, NEK7, NEK9, NIM1, NLK, OSR1, p38-alpha, p38-beta,p38-delta, p38-gamma, PAK1, PAK2, PAK3, PAK4, PAK6, PAK7, PCTK1, PCTK2,PCTK3, PDGFRA, PDGFRB, PDPK1, PFTAIRE2, PFTK1, PHKG1, PHKG2, PIK3C2B,PIK3C2G, PIK3CA, PIK3CA(C420R), PIK3CA(E542K), PIK3CA(E545A),PIK3CA(E545K), PIK3CA(H1047L), PIK3CA(H1047Y), PIK3CA(M1043I),PIK3CA(Q546K), PIK3CB, PIK3CD, PIK3CG, PIK4CB, PIM1, PIM2, PIM3,PIP5K1A, PIP5K2B, PKAC-ALPHA, PKAC-BETA, PKMYT1, PKN1, PKN2, PLK1, PLK2,PLK3, PLK4, PRKCD, PRKCE, PRKCH, PRKCQ, PRKD1, PRKD3, PRKG1, PRKG2,PRKR, PRKX, PRP4, PYK2, QSK, RAF1, RET, RET(M918T), RET(V804L),RET(V804M), RIOK1, RIOK2, RIOK3, RIPK1, RIPK2, RIPK4, ROCK1, ROCK2,ROS1, RPS6KA1(Kin.Dom.1-N-terminal), RPS6KA1(Kin.Dom.2-C-terminal),RPS6KA2(Kin.Dom.1-N-terminal), RPS6KA2(Kin.Dom.2-C-terminal),RPS6KA3(Kin.Dom.1-N-terminal), RPS6KA4(Kin.Dom.1-N-terminal),RPS6KA4(Kin.Dom.2-C-terminal), RPS6KA5(Kin.Dom.1-N-terminal),RPS6KA5(Kin.Dom.2-C-terminal), RPS6KA6(Kin.Dom.1-N-terminal),RPS6KA6(Kin.Dom.2-C-terminal), SBK1, SgK085, SgK110, SIK, SIK2, SLK,SNARK, SRC, SRMS, SRPK1, SRPK2, SRPK3, STK16, STK33, STK39, SYK, TAK1,TAO1, TAOK2, TAOK3, TBK1, TEC, TESK1, TGFBR1, TGFBR2, TIE1, TIE2, TLK1,TLK2, TNIK, TNK1, TNK2, TNNI3K, TRKA, TRKB, TRKC, TSSK1B, TTK, TXK,TYK2(JH1domain-catalytic), TYK2(JH2domain-pseudokinase), TYRO3, ULK1,ULK2, ULK3, VEGFR₂, WEE1, WEE2, YANK2, YANK3, YES, YSK1, YSK4, ZAK andZAP70. Compounds of the invention are screened against the kinase panel(wild type and/or mutation thereof) and inhibit the activity of at leastone of said panel members. In certain embodiments, the kinase isselected from ERK5, LRRK2, or EphA2.

Mutant forms of a kinase means single or multiple amino acid changesfrom the wild-type sequence.

The term “subject” as used herein refers to a mammal. A subjecttherefore refers to, for example, dogs, cats, horses, cows, pigs, guineapigs, and the like. Preferably the subject is a human. When the subjectis a human, the subject may be referred to herein as a patient.

Treat”, “treating” and “treatment” refer to a method of alleviating orabating a disease and/or its attendant symptoms.

As used herein, the term “pharmaceutically acceptable salt” refers tothose salts of the compounds formed by the process of the presentinvention which are, within the scope of sound medical judgment,suitable for use in contact with the tissues of humans and lower animalswithout undue toxicity, irritation, allergic response and the like, andare commensurate with a reasonable benefit/risk ratio. Pharmaceuticallyacceptable salts are well known in the art. For example, S. M. Berge, etal. describes pharmaceutically acceptable salts in detail in J.Pharmaceutical Sciences, 66: 1-19 (1977). The salts can be prepared insitu during the final isolation and purification of the compounds of theinvention, or separately by reacting the free base function with asuitable organic acid. Examples of pharmaceutically acceptable include,but are not limited to, nontoxic acid addition salts are salts of anamino group formed with inorganic acids such as hydrochloric acid,hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid orwith organic acids such as acetic acid, maleic acid, tartaric acid,citric acid, succinic acid or malonic acid or by using other methodsused in the art such as ion exchange. Other pharmaceutically acceptablesalts include, but are not limited to, adipate, alginate, ascorbate,aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate,camphorate, camphorsulfonate, citrate, cyclopentanepropionate,digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate,glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate,hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate,lactate, laurate, lauryl sulfate, malate, maleate, malonate,methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate,oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate,phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate,tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate salts,and the like. Representative alkali or alkaline earth metal saltsinclude sodium, lithium, potassium, calcium, magnesium, and the like.Further pharmaceutically acceptable salts include, when appropriate,nontoxic ammonium, quaternary ammonium, and amine cations formed usingcounterions such as halide, hydroxide, carboxylate, sulfate, phosphate,nitrate, alkyl having from 1 to 6 carbon atoms, sulfonate and arylsulfonate.

As used herein, the term “pharmaceutically acceptable ester” refers toesters of the compounds formed by the process of the present inventionwhich hydrolyze in vivo and include those that break down readily in thehuman body to leave the parent compound or a salt thereof. Suitableester groups include, for example, those derived from pharmaceuticallyacceptable aliphatic carboxylic acids, particularly alkanoic, alkenoic,cycloalkanoic and alkanedioic acids, in which each alkyl or alkenylmoiety advantageously has not more than 6 carbon atoms. Examples ofparticular esters include, but are not limited to, formates, acetates,propionates, butyrates, acrylates and ethylsuccinates.

The term “pharmaceutically acceptable prodrugs” as used herein refers tothose prodrugs of the compounds formed by the process of the presentinvention which are, within the scope of sound medical judgment,suitable for use in contact with the tissues of humans and lower animalswith undue toxicity, irritation, allergic response, and the like,commensurate with a reasonable benefit/risk ratio, and effective fortheir intended use, as well as the zwitterionic forms, where possible,of the compounds of the present invention. “Prodrug”, as used hereinmeans a compound which is convertible in vivo by metabolic means (e.g.by hydrolysis) to afford any compound delineated by the formulae of theinstant invention. Various forms of prodrugs are known in the art, forexample, as discussed in Bundgaard, (ed.), Design of Prodrugs, Elsevier(1985); Widder, et al. (ed.), Methods in Enzymology, vol. 4, AcademicPress (1985); Krogsgaard-Larsen, et al., (ed). “Design and Applicationof Prodrugs, Textbook of Drug Design and Development, Chapter 5, 113-191(1991); Bundgaard, et al., Journal of Drug Deliver Reviews, 8:1-38(1992); Bundgaard, J. of Pharmaceutical Sciences, 77:285 et seq. (1988);Higuchi and Stella (eds.) Prodrugs as Novel Drug Delivery Systems,American Chemical Society (1975); and Bernard Testa & Joachim Mayer,“Hydrolysis In Drug And Prodrug Metabolism: Chemistry, Biochemistry AndEnzymology,” John Wiley and Sons, Ltd. (2002).

This invention also encompasses pharmaceutical compositions containing,and methods of treating disorders through administering,pharmaceutically acceptable prodrugs of compounds of the invention. Forexample, compounds of the invention having free amino, amido, hydroxy orcarboxylic groups can be converted into prodrugs. Prodrugs includecompounds wherein an amino acid residue, or a polypeptide chain of twoor more (e.g., two, three or four) amino acid residues is covalentlyjoined through an amide or ester bond to a free amino, hydroxy orcarboxylic acid group of compounds of the invention. The amino acidresidues include but are not limited to the 20 naturally occurring aminoacids commonly designated by three letter symbols and also includes4-hydroxyproline, hydroxyysine, demosine, isodemosine,3-methylhistidine, norvalin, beta-alanine, gamma-aminobutyric acid,citrulline, homocysteine, homoserine, ornithine and methionine sulfone.Additional types of prodrugs are also encompassed. For instance, freecarboxyl groups can be derivatized as amides or alkyl esters. Freehydroxy groups may be derivatized using groups including but not limitedto hemisuccinates, phosphate esters, dimethylaminoacetates, andphosphoryloxymethyloxy carbonyls, as outlined in Advanced Drug DeliveryReviews, 1996, 19, 115. Carbamate prodrugs of hydroxy and amino groupsare also included, as are carbonate prodrugs, sulfonate esters andsulfate esters of hydroxy groups. Derivatization of hydroxy groups as(acyloxy)methyl and (acyloxy)ethyl ethers wherein the acyl group may bean alkyl ester, optionally substituted with groups including but notlimited to ether, amine and carboxylic acid functionalities, or wherethe acyl group is an amino acid ester as described above, are alsoencompassed. Prodrugs of this type are described in J. Med. Chem. 1996,39, 10. Free amines can also be derivatized as amides, sulfonamides orphosphonamides. All of these prodrug moieties may incorporate groupsincluding but not limited to ether, amine and carboxylic acidfunctionalities

Combinations of substituents and variables envisioned by this inventionare only those that result in the formation of stable compounds. Theterm “stable”, as used herein, refers to compounds which possessstability sufficient to allow manufacture and which maintains theintegrity of the compound for a sufficient period of time to be usefulfor the purposes detailed herein (e.g., therapeutic or prophylacticadministration to a subject).

Compounds of the Invention

In certain aspects, the invention provides a compound of formulae I-IX(or formulae A or F).

In one aspect, the invention provides a compound of formula I:

or a pharmaceutically acceptable salt, ester or prodrug thereof,

wherein,

A is a single bond or double bond;

R′ is H or alkyl;

L is absent, S, SO, SO₂, or CO;

X is an optional substituent (for example, halogen, —OH, —NO₂, —CN,—NH₂, protected amino, —NH—C₁-C₁₂-alkyl, —NH—C₂-C₁₂-alkenyl,—NH—C₂-C₁₂-alkenyl, —NH—C₃-C₁₂-cycloalkyl, —NH-aryl, —NH-heteroaryl,—NH-heterocycloalkyl, -dialkylamino, -diarylamino, -diheteroarylamino,—O—C₁-C₁₂-alkyl, —O—C₂-C₁₂-alkenyl, —O—C₂-C₁₂-alkenyl,—O—C₃-C₁₂-cycloalkyl, —O-aryl, —O-heteroaryl, —O-heterocycloalkyl,—C(O)—C₁-C₁₂-alkyl, —C(O)—C₂-C₁₂-alkenyl, —C(O)—C₂-C₁₂-alkenyl,—C(O)—C₃-C₁₂-cycloalkyl, —C(O)-aryl, —C(O)-heteroaryl,—C(O)-heterocycloalkyl, —CONH₂, —CONH—C₁-C₁₂-alkyl,—CONH—C₂-C₁₂-alkenyl, —CONH—C₂-C₁₂-alkenyl, —CONH—C₃-C₁₂-cycloalkyl,—CONH-aryl, —CONH-heteroaryl, —CONH-heterocycloalkyl,—OCO₂—C₁-C₁₂-alkyl, —OCO₂—C₂-C₁₂-alkenyl, —OCO₂—C₂-C₁₂-alkenyl,—OCO₂—C₃-C₁₂-cycloalkyl, —OCO₂-aryl, —OCO₂-heteroaryl,—OCO₂-heterocycloalkyl, —OCONH₂, —OCONH-—OCONH—C₂-C₁₂-alkenyl,—OCONH—C₂-C₁₂-alkenyl, —OCONH—C₃-C₁₂-cycloalkyl, —OCONH-aryl,—OCONH-heteroaryl, —OCONH-heterocycloalkyl, —NHC(O)—C₁-C₁₂-alkyl,—NHC(O)—C₂-C₁₂-alkenyl, —NHC(O)—C₂-C₁₂-alkenyl,—NHC(O)—C₃-C₁₂-cycloalkyl, —NHC(O)-aryl, —NHC(O)-heteroaryl,—NHC(O)-heterocycloalkyl, —NHCO₂—C₁-C₁₂-alkyl, —NHCO₂—C₂-C₁₂-alkenyl,—NHCO₂—C₂-C₁₂-alkenyl, —NHCO₂—C₃-C₁₂-cycloalkyl, —NHCO₂-aryl,—NHCO₂-heteroaryl, —NHCO₂-heterocycloalkyl, —NHC(O)NH₂,—NHC(O)NH—C₁-C₁₂-alkyl, —NHC(O)NH—C₂-C₁₂-alkenyl,—NHC(O)NH—C₂-C₁₂-alkenyl, —NHC(O)NH—C₃-C₁₂-cycloalkyl, —NHC(O)NH-aryl,—NHC(O)NH-heteroaryl, —NHC(O)NH-heterocycloalkyl, NHC(S)NH₂,—NHC(S)NH—C₁-C₁₂-alkyl, —NHC(S)NH—C₂-C₁₂-alkenyl,—NHC(S)NH—C₂-C₁₂-alkenyl, —NHC(S)NH—C₃-C₁₂-cycloalkyl, —NHC(S)NH-aryl,—NHC(S)NH-heteroaryl, —NHC(S)NH-heterocycloalkyl, —NHC(NH)NH₂,—NHC(NH)NH—C₁-C₁₂-alkyl, —NHC(NH)NH—C₂-C₁₂-alkenyl,—NHC(NH)NH—C₂-C₁₂-alkenyl, —NHC(NH)NH—C₃-C₁₂-cycloalkyl,—NHC(NH)NH-aryl, —NHC(NH)NH-heteroaryl, —NHC(NH)NH-heterocycloalkyl,—NHC(NH)—C₁-C₁₂-alkyl, —NHC(NH)—C₂-C₁₂-alkenyl, —NHC(NH)—C₂-C₁₂-alkenyl,—NHC(NH)—C₃-C₁₂-cycloalkyl, —NHC(NH)-aryl, —NHC(NH)-heteroaryl,—NHC(NH)-heterocycloalkyl, —C(NH)NH—C₁-C₁₂-alkyl,—C(NH)NH—C₂-C₁₂-alkenyl, —C(NH)NH—C₂-C₁₂-alkenyl,—C(NH)NH—C₃-C₁₂-cycloalkyl, —C(NH)NH-aryl, —C(NH)NH-heteroaryl,—C(NH)NH-heterocycloalkyl, —S(O)—C₁-C₁₂-alkyl, —S(O)—C₂-C₁₂-alkenyl,—S(O)—C₂-C₁₂-alkenyl, —S(O)—C₃-C₁₂-cycloalkyl, —S(O)-aryl,—S(O)-heteroaryl, —S(O)-heterocycloalkyl-SO₂NH₂, —SO₂NH—C₁-C₁₂-alkyl,—SO₂NH—C₂-C₁₂-alkenyl, —SO₂NH—C₂-C₁₂-alkenyl, —SO₂NH—C₃-C₁₂-cycloalkyl,—SO₂NH-aryl, —SO₂NH-heteroaryl, —SO₂NH-heterocycloalkyl,—NHSO₂—C₁-C₁₂-alkyl, —NHSO₂—C₂-C₁₂-alkenyl, —NHSO₂—C₂-C₁₂-alkenyl,—NHSO₂—C₃-C₁₂-cycloalkyl, —NHSO₂-aryl, —NHSO₂-heteroaryl,—NHSO₂-heterocycloalkyl, —CH₂NH₂, —CH₂SO₂CH₃, aryl, arylalkyl,heteroaryl, heteroarylalkyl, heterocycloalkyl, —C₃-C₁₂-cycloalkyl,polyalkoxyalkyl, polyalkoxy, -methoxymethoxy, -methoxyethoxy, —SH,—S—C₁-C₁₂-alkyl, —S—C₂-C₁₂-alkenyl, —S—C₂-C₁₂-alkenyl,—S—C₃-C₁₂-cycloalkyl, —S-aryl, —S-heteroaryl, —S-heterocycloalkyl, ormethylthiomethyl);

R₁ is H, alkyl, alkenyl, alkynyl, each containing 0, 1, 2, or 3heteroatoms selected from O, S, or N; or R₁ is aryl, arylalkyl,heteroaryl, heterocyclic, or carbocyclic; wherein R₁ may be optionallysubstituted;

R₂ is hydrogen, optionally substituted alkyl (including aralkyl),optionally substituted cycloalkyl, and optionally substitutedheterocyclyl; and

R₆ is hydrogen or optionally substituted alkyl.

In certain embodiments, R₁ is phenyl or pyridyl, each of which may beoptionally substituted.

In certain further embodiments, R₁ is substituted with 0-4 substituents,selected from N(R_(A))(R_(A)), C(O)NH(R_(A)), alkoxy, and heterocyclic,each of which may be further substituted; wherein each R_(A) isindependently selected from alkyl, and heterocyclic.

In certain further embodiments, R₁ is substituted with 0-4 substituents,

selected from alkoxy,

In certain embodiments, R₂ is H, methyl, or ethyl.

In certain embodiments, R₆ is H.

In certain embodiments, X is H.

In certain embodiments, R′ is H.

In certain embodiments, L is absent.

In certain embodiments, A is a single bond.

In another aspect, the invention provides a compound of formula II:

or a pharmaceutically acceptable salt, ester or prodrug thereof,

wherein,

R′ is H or alkyl;

L is absent, S, SO, SO₂, or CO;

X is an optional substituent as defined for formula I;

E is NR₂ or CHR₂;

R₁ is H, alkyl, alkenyl, alkynyl, each containing 0, 1, 2, or 3heteroatoms selected from O, S, or N; or R₁ is aryl, arylalkyl,heteroaryl, heterocyclic, or carbocyclic; wherein R₁ may be optionallysubstituted;

R₂ is, independently for each occurrence, hydrogen, optionallysubstituted alkyl (including aralkyl), optionally substitutedcycloalkyl, and optionally substituted heterocyclyl; and

R₆ is hydrogen or optionally substituted alkyl.

In certain embodiments, R₁ is phenyl or pyridyl, each of which may beoptionally substituted.

In certain further embodiments, R₁ is substituted with 0-4 substituents,selected from N(R_(A))(R_(A)), C(O)NH(R_(A)), alkoxy, and heterocyclic,each of which may be further substituted; wherein each R_(A) isindependently selected from alkyl, and heterocyclic.

In certain further embodiments, R₁ is substituted with 0-4 substituents,

selected from alkoxy,

In certain embodiments, E is NR₂.

In certain embodiments, R₂ is H, methyl, or ethyl. In certainembodiments, each R₂ is H, methyl, optionally substituted benzyl,cyclopentyl, or pyranyl. In certain embodiments, if E is NR₂, then oneR₂ is H or methyl, and the other R₂ is H, methyl, optionally substitutedbenzyl, cyclopentyl, or pyranyl. In certain embodiments, the optionallysubstituted benzyl is 2-chlorobenzyl, 2,4-dichlorobenzyl, 2-chloro,4-fluorobenzyl, or 2-methylbenzyl.

In certain embodiments, R₆ is H.

In certain embodiments, X is H.

In certain embodiments, R′ is H.

In certain embodiments, L is absent.

In another aspect, the invention provides a compound of formula III:

or a pharmaceutically acceptable salt, ester or prodrug thereof,

wherein,

R′ is H or alkyl;

L is absent, S, SO, SO₂, or CO;

X is an optional substituent as defined for formula I;

R₁ is H, alkyl, alkenyl, alkynyl, each containing 0, 1, 2, or 3heteroatoms selected from O, S, or N; or R₁ is aryl, arylalkyl,heteroaryl, heterocyclic, or carbocyclic; wherein R₁ may be optionallysubstituted;

R₂ is, independently for each occurrence, hydrogen, optionallysubstituted alkyl (including aralkyl), optionally substitutedcycloalkyl, and optionally substituted heterocyclyl; and

R₆ is hydrogen or optionally substituted alkyl.

In certain embodiments, R₁ is phenyl or pyridyl, each of which may beoptionally substituted.

In certain further embodiments, R₁ is substituted with 0-4 substituents,selected from N(R_(A))(R_(A)), C(O)NH(R_(A)), alkoxy, and heterocyclic,each of which may be further substituted; wherein each R_(A) isindependently selected from alkyl, and heterocyclic.

In certain further embodiments, R₁ is substituted with 0-4 substituents,selected from alkoxy,

In certain embodiments, R₂ is H, methyl, or ethyl.

In certain embodiments, R₆ is H.

In certain embodiments, X is H.

In certain embodiments, R′ is H.

In certain embodiments, L is absent.

In another aspect, the invention provides a compound of formula IV:

or a pharmaceutically acceptable salt, ester or prodrug thereof,

wherein,

R′ is H or alkyl;

L is absent, S, SO, SO₂, or CO;

X is an optional substituent as defined for formula I;

R₁ is H, alkyl, alkenyl, alkynyl, each containing 0, 1, 2, or 3heteroatoms selected from O, S, or N; or R₁ is aryl, arylalkyl,heteroaryl, heterocyclic, or carbocyclic; wherein R₁ may be optionallysubstituted;

R₂ is, independently for each occurrence, hydrogen, optionallysubstituted alkyl (including aralkyl), optionally substitutedcycloalkyl, and optionally substituted heterocyclyl; and

R₆ is hydrogen or optionally substituted alkyl.

In certain embodiments, R₁ is phenyl or pyridyl, each of which may beoptionally substituted.

In certain further embodiments, R₁ is substituted with 0-4 substituents,selected from N(R_(A))(R_(A)), C(O)NH(R_(A)), alkoxy, and heterocyclic,each of which may be further substituted; wherein each R_(A) isindependently selected from alkyl, and heterocyclic.

In certain further embodiments, R₁ is substituted with 0-4 substituents,

selected from alkoxy,

In certain embodiments, R₂ is H, methyl, or ethyl.

In certain embodiments, R₆ is H.

In certain embodiments, X is H.

In certain embodiments, R′ is H.

In certain embodiments, L is absent.

In another aspect, the invention provides a compound of formula V:

or a pharmaceutically acceptable salt, ester or prodrug thereof,

wherein,

X is an optional substituent as defined for formula I;

R₂ is hydrogen or optionally substituted alkyl;

R₃ is —OH or —O-(optionally substituted alkyl);

R₄ is hydrogen or optionally substituted alkyl; and

R₆ is hydrogen or optionally substituted alkyl.

In certain embodiments, R₂ is H, methyl, or ethyl.

R₃ is —OCH₃ or —OCH₂CH₃.

In certain embodiments, X is H.

In certain embodiments, R′ is H.

In certain embodiments, R₄ is methyl or ethyl.

In another aspect, the invention provides a compound of Formula VI:

or a pharmaceutically acceptable salt, ester or prodrug thereof,wherein,

R′ is H or alkyl;

L is absent, S, SO, SO₂, or CO;

X is an optional substituent as defined for formula I;

R₁ is H, alkyl, alkenyl, alkynyl, each containing 0, 1, 2, or 3heteroatoms selected from O, S, or N; or R₁ is aryl, arylalkyl,heteroaryl, heterocyclic, or carbocyclic; wherein R₁ may be optionallysubstituted;

R₂ is, independently for each occurrence, hydrogen, optionallysubstituted alkyl, optionally substituted cycloalkyl, and optionallysubstituted heterocyclyl; or

two X moieties on adjacent atoms of the thiophene ring can form,together with the atoms to which they are attached, a phenyl ring; and

R₆ is hydrogen or optionally substituted alkyl.

In another aspect, the invention provides a compound of Formula VII:

or a pharmaceutically acceptable salt, ester or prodrug thereof,wherein,

R′ is H or alkyl;

L is absent, S, SO, SO₂, or CO;

X is an optional substituent as defined for formula I;

R₁ is H, alkyl, alkenyl, alkynyl, each containing 0, 1, 2, or 3heteroatoms selected from O, S, or N; or R₁ is aryl, arylalkyl,heteroaryl, heterocyclic, or carbocyclic; wherein R₁ may be optionallysubstituted;

R₂ is hydrogen, optionally substituted alkyl, optionally substitutedcycloalkyl, and optionally substituted heterocyclyl; and

R₆ is hydrogen or optionally substituted alkyl.

In another aspect, the invention provides a compound of Formula VIII:

or a pharmaceutically acceptable salt, ester or prodrug thereof,wherein,

R′ is H or alkyl;

L is absent, S, SO, SO₂, or CO;

X is an optional substituent as defined for formula I;

Z is O or S;

R₁ is H, alkyl, alkenyl, alkynyl, each containing 0, 1, 2, or 3heteroatoms selected from O, S, or N; or R₁ is aryl, arylalkyl,heteroaryl, heterocyclic, or carbocyclic; wherein R₁ may be optionallysubstituted;

R₂ is hydrogen, optionally substituted alkyl, optionally substitutedcycloalkyl, and optionally substituted heterocyclyl; and

R₆ is hydrogen or optionally substituted alkyl.

In another aspect, the invention provides a compound of Formula IX:

or a pharmaceutically acceptable salt, ester or prodrug thereof,wherein,

A is a single bond or double bond;

R′ is H or alkyl;

L is absent, S, SO, SO₂, or CO;

Y is hydrogen, optionally substituted alkyl, optionally substitutedcycloalkyl, and optionally substituted heterocyclyl;

R₁ is H, alkyl, alkenyl, alkynyl, each containing 0, 1, 2, or 3heteroatoms selected from O, S, or N; or R₁ is aryl, arylalkyl,heteroaryl, heterocyclic, or carbocyclic; wherein R₁ may be optionallysubstituted;

R₂ and R₂′ are each independently hydrogen, optionally substitutedalkyl, optionally substituted cycloalkyl, and optionally substitutedheterocyclyl; or Y and R₂′ can form, together with the atoms to whichthey are attached, a five-membered ring; and

R₆ is hydrogen or optionally substituted alkyl.

In one aspect, the invention provides a compound of formula A:

or a pharmaceutically acceptable salt, ester or prodrug thereof,

-   -   wherein,        -   X is CHR₄, CR₄, NH, NR₄ or N;        -   Y is NR₅, N, S, SO, SO₂, O, CHR₅, or CR₅; wherein at least            one of X and Y is NH, NR₄, NR₅, N, S, SO, SO₂, or O;        -   A is a single bond or double bond;        -   B is a single bond or double bond, wherein both A and B are            not double bonds;    -   R′ is H or alkyl;    -   L is absent, S, SO, SO₂, or CO;    -   R₁ is H, alkyl, alkenyl, alkynyl, each containing 0, 1, 2, or 3        heteroatoms selected from O, S, or N; or R₁ is aryl, arylalkyl,        heteroaryl, heterocyclic, or carbocyclic; wherein R₁ may be        optionally substituted;    -   R₂ is hydrogen or optionally substituted alkyl;    -   R₃ is hydrogen, alkyl, alkenyl, aryl, arylalkyl, heteroaryl,        heterocyclic, or carbocyclic, each of which may be optionally        substituted;    -   R₄ is hydrogen, alkyl, alkenyl, aryl, arylalkyl, heteroaryl,        heterocyclic, or carbocyclic, each of which may be optionally        substituted;    -   R₅ is hydrogen, alkyl, alkenyl, aryl, arylalkyl, heteroaryl,        heterocyclic, or carbocyclic, each of which may be optionally        substituted;    -   or R₃ and X, together with the atoms to which they are attached,        form a 3-8 membered carbocyclic, aryl, heterocyclic, or        heteroaryl; each of which is optionally substituted;    -   or X and Y, together with the atoms to which they are attached,        form a 3-8 membered carbocyclic, aryl, heterocyclic, or        heteroaryl; each of which is optionally substituted; and    -   R₆ is hydrogen or optionally substituted alkyl.

In another aspect, the invention provides a compound of formula F:

or a pharmaceutically acceptable salt, ester or prodrug thereof,

wherein,

R¹ is alkyl, aryl, heteroaryl, heterocyclic, or carbocyclic, wherein R1may be optionally substituted;

R² is hydrogen or optionally substituted alkyl;

R₅ is hydrogen, optionally substituted alkyl, optionally substitutedaralkyl, or optionally substituted carbocyclic; and

R₆ is hydrogen or optionally substituted alkyl;

each R₇ is independently alkyl, alkenyl, aryl, arylalkyl, heteroaryl,heterocyclic, carbocyclic, alkoxy, NH(alkyl), NH(aryl), N(alkyl)(alkyl),or N(alkyl)(aryl), each of which may be optionally substituted; hal,nitro, or cyano; and p is 0-4;

or, when p is 2, 3, or 4, two occurrences of R₇ may be joined, togetherwith the carbon atoms to which they are attached, to form an aryl orheteroaryl ring (e.g., having 5 or 6 atoms in the aryl or heteroarylring), e.g., a fused phenyl ring.

In certain embodiments, R₁ is methyl, ethyl, propyl, iso-propyl, butyl,s-butyl, t-butyl, pentyl, hexyl, cyclohexyl, piperidinyl, pyrrolidino,phenyl, 1-naphthyl, 2-naphthyl, pyridyl, pyrimidinyl, pyrazinyl,pyridizinyl, quinolinyl, thienyl, thiazolyl, oxazolyl, isoxazolyl,pyrrolyl, furanyl, isoquinolinyl, imiazolyl, or triazolyl, each of whichmay be optionally substituted.

In a further embodiment, R₁ is phenyl or pyridyl, each of which may beoptionally substituted. In a further embodiment, R₁ is pyrazolyl. Incertain embodiments, R₁ is

In another embodiment, R₁ is substituted with 0-4 substituents, selectedfrom hal, nitro, cyano, hydroxyl, amino, NH(RA), N(RA)(RA), CO₂H,C(O)RA, C(O)ORA, C(O)NH₂, C(O)NH(RA), C(O)N(RA)(RA),alkyl, aryl,arylalkyl, alkoxy, heteroaryl, heterocyclic, and carbocyclic, each ofwhich may be further substituted; wherein each R_(A) is independentlyselected from alkyl, carbocyclic, aryl, heteroaryl, and heterocyclic.

In certain embodiments, two occurrences of R₇ are joined to form aphenyl ring.

In certain embodiments, the compound is:

Representative compounds of the invention include, but are not limitedto, the following compounds of Tables 1-6, which follow the Examples.

Suitable syntheses for compounds of the invention can be found in theExamples below. In addition, syntheses disclosed, e.g., in U.S. PatentApplication Publication No. US-2012-0040961-A1 (the contents of which isincorporated herein by reference), can be used, with appropriatemodifications, to prepare compounds of the invention.

Another embodiment is a method of making a compound of any of theformulae herein using any one, or combination of, reactions delineatedherein. The method can include the use of one or more intermediates orchemical reagents delineated herein.

Another aspect is an isotopically labeled compound of any of theformulae delineated herein. Such compounds have one or more isotopeatoms which may or may not be radioactive (e.g., ³H, ²H, ¹⁴C, ¹³C, ³⁵S,³²P, ¹²⁵I, and ¹³¹I) introduced into the compound. Such compounds areuseful for drug metabolism studies and diagnostics, as well astherapeutic applications.

A compound of the invention can be prepared as a pharmaceuticallyacceptable acid addition salt by reacting the free base form of thecompound with a pharmaceutically acceptable inorganic or organic acid.Alternatively, a pharmaceutically acceptable base addition salt of acompound of the invention can be prepared by reacting the free acid formof the compound with a pharmaceutically acceptable inorganic or organicbase.

Alternatively, the salt forms of the compounds of the invention can beprepared using salts of the starting materials or intermediates.

The free acid or free base forms of the compounds of the invention canbe prepared from the corresponding base addition salt or acid additionsalt from, respectively. For example a compound of the invention in anacid addition salt form can be converted to the corresponding free baseby treating with a suitable base (e.g., ammonium hydroxide solution,sodium hydroxide, and the like). A compound of the invention in a baseaddition salt form can be converted to the corresponding free acid bytreating with a suitable acid (e.g., hydrochloric acid, etc.).

Prodrug derivatives of the compounds of the invention can be prepared bymethods known to those of ordinary skill in the art (e.g., for furtherdetails see Saulnier et al., (1994), Bioorganic and Medicinal ChemistryLetters, Vol. 4, p. 1985). For example, appropriate prodrugs can beprepared by reacting a non-derivatized compound of the invention with asuitable carbamylating agent (e.g., 1,1-acyloxyalkylcarbanochloridate,para-nitrophenyl carbonate, or the like).

Protected derivatives of the compounds of the invention can be made bymeans known to those of ordinary skill in the art. A detaileddescription of techniques applicable to the creation of protectinggroups and their removal can be found in T. W. Greene, “ProtectingGroups in Organic Chemistry”, 3rd edition, John Wiley and Sons, Inc.,1999.

Compounds of the present invention can be conveniently prepared, orformed during the process of the invention, as solvates (e.g.,hydrates). Hydrates of compounds of the present invention can beconveniently prepared by recrystallization from an aqueous/organicsolvent mixture, using organic solvents such as dioxin, tetrahydrofuranor methanol.

Acids and bases useful in the methods herein are known in the art. Acidcatalysts are any acidic chemical, which can be inorganic (e.g.,hydrochloric, sulfuric, nitric acids, aluminum trichloride) or organic(e.g., camphorsulfonic acid, p-toluenesulfonic acid, acetic acid,ytterbium triflate) in nature. Acids are useful in either catalytic orstoichiometric amounts to facilitate chemical reactions. Bases are anybasic chemical, which can be inorganic (e.g., sodium bicarbonate,potassium hydroxide) or organic (e.g., triethylamine, pyridine) innature. Bases are useful in either catalytic or stoichiometric amountsto facilitate chemical reactions.

In addition, some of the compounds of this invention have one or moredouble bonds, or one or more asymmetric centers. Such compounds canoccur as racemates, racemic mixtures, single enantiomers, individualdiastereomers, diastereomeric mixtures, and cis- or trans- or E- orZ-double isomeric forms, and other stereoisomeric forms that may bedefined, in terms of absolute stereochemistry, as (R)- or (S)-, or as(D)- or (L)-for amino acids. All such isomeric forms of these compoundsare expressly included in the present invention. Optical isomers may beprepared from their respective optically active precursors by theprocedures described above, or by resolving the racemic mixtures. Theresolution can be carried out in the presence of a resolving agent, bychromatography or by repeated crystallization or by some combination ofthese techniques which are known to those skilled in the art. Furtherdetails regarding resolutions can be found in Jacques, et al.,Enantiomers, Racemates, and Resolutions (John Wiley & Sons, 1981). Thecompounds of this invention may also be represented in multipletautomeric forms, in such instances, the invention expressly includesall tautomeric forms of the compounds described herein (e.g., alkylationof a ring system may result in alkylation at multiple sites, theinvention expressly includes all such reaction products). When thecompounds described herein contain olefinic double bonds or othercenters of geometric asymmetry, and unless specified otherwise, it isintended that the compounds include both E and Z geometric isomers.Likewise, all tautomeric forms are also intended to be included. Theconfiguration of any carbon-carbon double bond appearing herein isselected for convenience only and is not intended to designate aparticular configuration unless the text so states; thus a carbon-carbondouble bond depicted arbitrarily herein as trans may be cis, trans, or amixture of the two in any proportion. All such isomeric forms of suchcompounds are expressly included in the present invention. All crystalforms of the compounds described herein are expressly included in thepresent invention.

The synthesized compounds can be separated from a reaction mixture andfurther purified by a method such as column chromatography, highpressure liquid chromatography, or recrystallization. As can beappreciated by the skilled artisan, further methods of synthesizing thecompounds of the formulae herein will be evident to those of ordinaryskill in the art. Additionally, the various synthetic steps may beperformed in an alternate sequence or order to give the desiredcompounds. In addition, the solvents, temperatures, reaction durations,etc. delineated herein are for purposes of illustration only and one ofordinary skill in the art will recognize that variation of the reactionconditions can produce the desired bridged macrocyclic products of thepresent invention. Synthetic chemistry transformations and protectinggroup methodologies (protection and deprotection) useful in synthesizingthe compounds described herein are known in the art and include, forexample, those such as described in R. Larock, Comprehensive OrganicTransformations, VCH Publishers (1989); T. W. Greene and P. G. M. Wuts,Protective Groups in Organic Synthesis, 2d. Ed., John Wiley and Sons(1991); L. Fieser and M. Fieser, Fieser and Fieser's Reagents forOrganic Synthesis, John Wiley and Sons (1994); and L. Paquette, ed.,Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons(1995), and subsequent editions thereof.

The compounds of this invention may be modified by appending variousfunctionalities via any synthetic means delineated herein to enhanceselective biological properties. Such modifications are known in the artand include those which increase biological penetration into a givenbiological system (e.g., blood, lymphatic system, central nervoussystem), increase oral availability, increase solubility to allowadministration by injection, alter metabolism and alter rate ofexcretion.

The compounds of the invention are defined herein by their chemicalstructures and/or chemical names. Where a compound is referred to byboth a chemical structure and a chemical name, and the chemicalstructure and chemical name conflict, the chemical structure isdeterminative of the compound's identity.

The recitation of a listing of chemical groups in any definition of avariable herein includes definitions of that variable as any singlegroup or combination of listed groups. The recitation of an embodimentfor a variable herein includes that embodiment as any single embodimentor in combination with any other embodiments or portions thereof.

Methods of the Invention

In another aspect, the invention provides a method of treating a diseasein a subject comprising administering to the subject a compound,pharmaceutically acceptable salt, ester or prodrug of formulae I-IX (orformulae A or F).

In one embodiment, the invention provides a method wherein the diseaseis mediated by a kinase selected from a MAP kinase, a mitotic spindlekinase, and a polo kinase.

In another embodiment, the invention provides a method wherein thedisease is mediated by a kinase selected from MPS1, ERK5, BMK1, MAPK7,polo kinase 1, 2, 3, or 4, Ack1, Ack2, Abl, DCAMKL1, ABL1, Abl mutants,DCAMKL2, ARK5, BRK, MKNK2, FGFR4, TNK1, PLK1, ULK2, PLK4, PRKD1, PRKD2,PRKD3, ROS1, RPS6KA6, TAOK1, TAOK3, TNK2, Bcr-Abl, GAK, cSrc, TPR-Met,Tie2, MET, FGFR3, Aurora, Axl, Bmx, BTK, c-kit, CHK2, Flt3, MST2,p70S6K, PDGFR, PKB, PKC, Raf, ROCK-H, Rsk1, SGK, TrkA, TrkB and TrkC. Ina further embodiment, the kinase is ERK-5, MPS1, or BMK-1. In a furtherembodiment, the kinase is ERK-5, LRKK2, or EphA2.

In another embodiment, the invention provides a method wherein thedisease is cancer or a proliferation disease.

In a further embodiment, the disease is lung, colon, breast, prostate,liver, pancreas, brain, kidney, ovaries, stomach, skin, and bonecancers, gastric, breast, pancreatic cancer, glioma, and hepatocellularcarcinoma, papillary renal carcinoma, head and neck squamous cellcarcinoma, leukemias, lymphomas, myelomas, and solid tumors.

In another embodiment, the disease is inflammation, arthritis,rheumatoid arthritis, spondylarthropathies, gouty arthritis,osteoarthritis, juvenile arthritis, and other arthritic conditions,systemic lupus erthematosus (SLE), skin-related conditions, psoriasis,eczema, burns, dermatitis, neuroinflammation, allergy, pain, neuropathicpain, fever, pulmonary disorders, lung inflammation, adult respiratorydistress syndrome, pulmonary sarcoisosis, asthma, silicosis, chronicpulmonary inflammatory disease, and chronic obstructive pulmonarydisease (COPD), cardiovascular disease, arteriosclerosis, myocardialinfarction (including post-myocardial infarction indications),thrombosis, congestive heart failure, cardiac reperfusion injury, aswell as complications associated with hypertension and/or heart failuresuch as vascular organ damage, restenosis, cardiomyopathy, strokeincluding ischemic and hemorrhagic stroke, reperfusion injury, renalreperfusion injury, ischemia including stroke and brain ischemia, andischemia resulting from cardiac/coronary bypass, neurodegenerativedisorders, liver disease and nephritis, gastrointestinal conditions,inflammatory bowel disease, Crohn's disease, gastritis, irritable bowelsyndrome, ulcerative colitis, ulcerative diseases, gastric ulcers, viraland bacterial infections, sepsis, septic shock, gram negative sepsis,malaria, meningitis, HIV infection, opportunistic infections, cachexiasecondary to infection or malignancy, cachexia secondary to acquiredimmune deficiency syndrome (AIDS), AIDS, ARC (AIDS related complex),pneumonia, herpes virus, myalgias due to infection, influenza,autoimmune disease, graft vs. host reaction and allograft rejections,treatment of bone resorption diseases, osteoporosis, multiple sclerosis,cancer, leukemia, lymphoma, colorectal cancer, brain cancer, bonecancer, epithelial call-derived neoplasia (epithelial carcinoma), basalcell carcinoma, adenocarcinoma, gastrointestinal cancer, lip cancer,mouth cancer, esophageal cancer, small bowel cancer, stomach cancer,colon cancer, liver cancer, bladder cancer, pancreas cancer, ovariancancer, cervical cancer, lung cancer, breast cancer, skin cancer,squamus cell and/or basal cell cancers, prostate cancer, renal cellcarcinoma, and other known cancers that affect epithelial cellsthroughout the body, chronic myelogenous leukemia (CML), acute myeloidleukemia (AML) and acute promyelocytic leukemia (APL), angiogenesisincluding neoplasia, metastasis, central nervous system disorders,central nervous system disorders having an inflammatory or apoptoticcomponent, Alzheimer's disease, Parkinson's disease, Huntington'sdisease, amyotrophic lateral sclerosis, spinal cord injury, andperipheral neuropathy, Canine B-Cell Lymphoma.

In a further embodiment, the disease is inflammation, arthritis,rheumatoid arthritis, spondylarthropathies, gouty arthritis,osteoarthritis, juvenile arthritis, and other arthritic conditions,systemic lupus erthematosus (SLE), skin-related conditions, psoriasis,eczema, dermatitis, pain, pulmonary disorders, lung inflammation, adultrespiratory distress syndrome, pulmonary sarcoisosis, asthma, chronicpulmonary inflammatory disease, and chronic obstructive pulmonarydisease (COPD), cardiovascular disease, arteriosclerosis, myocardialinfarction (including post-myocardial infarction indications),congestive heart failure, cardiac reperfusion injury, inflammatory boweldisease, Crohn's disease, gastritis, irritable bowel syndrome, leukemia,lymphoma.

In another aspect, the invention provides a method of treating a kinasemediated disorder in a subject comprising: administering to the subjectidentified as in need thereof a compound, pharmaceutically acceptablesalt, ester or prodrug of formulae I-IX (or formulae A or F).

In one embodiment, the compound is an inhibitor of MPS1, ERK5, BMK1,MAPK7, polo kinase 1, 2, 3, or 4, Ack1, Ack2, Abl, DCAMKL1, ABL1, Ablmutants, DCAMKL2, ARK5, BRK, MKNK2, FGFR4, TNK1, PLK1, ULK2, PLK4,PRKD1, PRKD2, PRKD3, ROS1, RPS6KA6, TAOK1, TAOK3, TNK2, Bcr-Abl, GAK,cSrc, TPR-Met, Tie2, MET, FGFR3, Aurora, Axl, Bmx, BTK, c-kit, CHK2,Flt3, MST2, p70S6K, PDGFR, PKB, PKC, Raf, ROCK-H, Rsk1, SGK, TrkA, TrkBor TrkC. In a further embodiment, the compound is an inhibitor of ERK-5,MPS1, or BMK-1. In a further embodiment, the kinase is ERK-5, LRKK2, orEphA2.

In certain embodiments, the subject is administered an additionaltherapeutic agent.

In a further embodiment, the compound and the additional therapeuticagent are administered simultaneously or sequentially.

In another aspect, the invention provides a method for reducingkinase-dependent cell growth comprising contacting a cell with a kinaseinhibitor compound of formulae I-IX (or formulae A or F).

In other aspects, the invention provides a method of inhibiting kinasein a subject identified as in need of such treatment, comprisingadministering a compound of formulae I-IX (or formulae A or F).

In certain embodiments, the invention provides a method wherein thesubject is a human.

In other embodiments, the invention provides a method wherein the kinaseinhibitor has a Ki for inhibiting kinase less than about 1 micromolar.

In one embodiment, the invention provides a method of synthesizing acompound of formulae I-IX (or formulae A or F).

Another aspect of this invention provides compounds or compositions thatare inhibitors of protein kinases, and thus are useful for the treatmentof the diseases, disorders, and conditions, along with other usesdescribed herein. In certain embodiments, these compositions optionallyfurther comprise one or more additional therapeutic agents.

The present invention provides compounds and compositions that areuseful as inhibitors of protein kinases. In certain embodiments, theinvention provides compounds and compositions that are useful asinhibitors of protein kinases selected from AAK1, ABL1, ABL1(E255K),ABL1(F317I), ABL1(F317L), ABL1(H396P), ABL1(M351T), ABL1(Q252H),ABL1(T3151), ABL1(Y253F), ABL2, ACVR1, ACVR1B, ACVR2A, ACVR2B, ACVRL1,ADCK3, ADCK4, AKT1, AKT2, AKT3, ALK, AMPK-alpha1, AMPK-alpha2, ANKK1,ARK5, ASK1, ASK2, AURKA, AURKB, AURKC, AXL, BIKE, BLK, BMPR1A, BMPR1B,BMPR2, BMX, BRAF, BRAF(V600E), BRK, BRSK1, BRSK2, BTK, CAMK1, CAMK1D,CAMK1G, CAMK2A, CAMK2D, CAMK2G, CAMK4, CAMKK1, CAMKK2, CDC2L1, CDC2L2,CDK11, CDK2, CDK3, CDK5, CDK7, CDK8, CDK9, CDKL2, CDKL3, CDKL5, CHECK1,CHEK2, CIT, CLK1, CLK2, CLK3, CLK4, CSF1R, CSK, CSNK1A1L, CSNK1D,CSNK1E, CSNK1G1, CSNK1G3, CSNK2A1, CSNK2A2, CTK, DAPK1, DAPK2, DAPK3,DCAMKL1, DCAMKL2, DCAMKL3, DDR1, DDR2, DLK, DMPK, DMPK2, DRAK1, DRAK2,DYRK1A, DYRK1B, DYRK2, EGFR, EGFR (E746-A750DEL), EGFR (G719C), EGFR(G719S), EGFR(L747-E749del, A750P), EGFR(L747-S752del, P753S),EGFR(L747-T751del,Sins), EGFR(L858R), EGFR(L858R,T790M), EGFR(L861Q),EGFR(5752-1759del), EPHA1, EPHA2, EPHA3, EPHA4, EPHA5, EPHA6, EPHA7,EPHA8, EPHB1, EPHB2, EPHB3, EPHB4, EPHB6, ERBB2, ERBB3, ERBB4, ERK1,ERK2, ERK3, ERK4, ERK5, ERK8, ERN1, FAK, FER, FES, FGFR1, FGFR2, FGFR3,FGFR3(G697C), FGFR4, FGR, FLT1, FLT3, FLT3(D835H), FLT3(D835Y),FLT3(ITD), FLT3(K663Q), FLT3(N841I), FLT4, FRK, FYN, GAK,GCN2(Kin.Dom.2,S808G), GRK1, GRK4, GRK7, GSK3A, GSK3B, HCK, HIPK1,HIPK2, HIPK3, HIPK4, HPK1, HUNK, ICK, IGF1R, IKK-ALPHA, IKK-BETA,IKK-EPSILON, INSR, INSRR, IRAK1, IRAK3, ITK, JAK1(JH1domain-catalytic),JAK1(JH2domain-pseudokinase), JAK2(JH1domain-catalytic),JAK3(JH1domain-catalytic), JNK1, JNK2, JNK3, KIT, KIT(D816V),KIT(L576P), KIT(V559D), KIT(V559D,T670I), KIT(V559D,V654A), LATS1,LATS2, LCK, LIMK1, LIMK2, LKB1, LOK, LTK, LYN, LZK, MAK, MAP3K1,MAP2K15, MAP3K2, MAP3K3, MAP3K4, MAP4K2, MAP4K3, MAP4K5, MAPKAPK2,MAPKAPK5, MARK1, MARK2, MARK3, MARK4, MAST1, MEK1, MEK2, MEK3, MEK4,MEK6, MELK, MERTK, MET, MET(M1250T), MET(Y1235D), MINK, MKNK1, MKNK2,MLCK, MLK1, MLK2, MLK3, MRCKA, MRCKB, MST1, MST1R, MST2, MST3, MST4,MUSK, MYLK, MYLK2, MYO3A, MYO3B, NDR1, NDR2, NEK1, NEK2, NEK5, NEK6,NEK7, NEK9, NIM1, NLK, OSR1, p38-alpha, p38-beta, p38-delta, p38-gamma,PAK1, PAK2, PAK3, PAK4, PAK6, PAK7, PCTK1, PCTK2, PCTK3, PDGFRA, PDGFRB,PDPK1, PFTAIRE2, PFTK1, PHKG1, PHKG2, PIK3C2B, PIK3C2G, PIK3CA,PIK3CA(C420R), PIK3CA(E542K), PIK3CA(E545A), PIK3CA(E545K),PIK3CA(H1047L), PIK3CA(H1047Y), PIK3CA(M1043I), PIK3CA(Q546K), PIK3CB,PIK3CD, PIK3CG, PIK4CB, PIM1, PIM2, PIM3, PIP5K1A, PIP5K2B, PKAC-ALPHA,PKAC-BETA, PKMYT1, PKN1, PKN2, PLK1, PLK2, PLK3, PLK4, PRKCD, PRKCE,PRKCH, PRKCQ, PRKD1, PRKD3, PRKG1, PRKG2, PRKR, PRKX, PRP4, PYK2, QSK,RAF1, RET, RET(M918T), RET(V804L), RET(V804M), RIOK1, RIOK2, RIOK3,RIPK1, RIPK2, RIPK4, ROCK1, ROCK2, ROS1, RPS6KA1(Kin.Dom.1-N-terminal),RPS6KA1(Kin.Dom.2-C-terminal), RPS6KA2(Kin.Dom.1-N-terminal),RPS6KA2(Kin.Dom.2-C-terminal), RPS6KA3(Kin.Dom.1-N-terminal),RPS6KA4(Kin.Dom.1-N-terminal), RP S6KA4(Kin.Dom.2-C-terminal),RPS6KA5(Kin.Dom.1-N-terminal), RPS6KA5(Kin.Dom.2-C-terminal),RPS6KA6(Kin.Dom.1-N-terminal), RPS6KA6(Kin.Dom.2-C-terminal), SBK1,SgK085, SgK110, SIK, SIK2, SLK, SNARK, SRC, SRMS, SRPK1, SRPK2, SRPK3,STK16, STK33, STK39, SYK, TAK1, TAO1, TAOK2, TAOK3, TBK1, TEC, TESK1,TGFBR1, TGFBR2, TIE1, TIE2, TLK1, TLK2, TNIK, TNK1, TNK2, TNNI3K, TRKA,TRKB, TRKC, TSSK1B, TTK, TXK, TYK2(JH1domain-catalytic),TYK2(JH2domain-pseudokinase), TYRO3, ULK1, ULK2, ULK3, VEGFR₂, WEE1,WEE2, YANK2, YANK3, YES, YSK1, YSK4, ZAK and ZAP70. In a furtherembodiment, the kinase is ERK-5, LRKK2, or EphA2.

In some embodiments, the present invention provides compounds andcompositions that are useful as inhibitors of protein kinases selectedfrom MPS1, ERK5, BMK1, MAPK7, polo kinase 1, 2, 3, or 4, Ack1, Ack2,Abl, DCAMKL1, ABL1, Abl mutants, DCAMKL2, ARK5, BRK, MKNK2, FGFR4, TNK1,PLK1, ULK2, PLK4, PRKD1, PRKD2, PRKD3, ROS1, RPS6KA6, TAOK1, TAOK3,TNK2, Bcr-Abl, GAK, cSrc, TPR-Met, Tie2, MET, FGFR3, Aurora, Axl, Bmx,BTK, c-kit, CHK2, Flt3, MST2, p70S6K, PDGFR, PKB, PKC, Raf, ROCK-H,Rsk1, SGK, TrkA, TrkB and TrkC. In a further embodiment, the kinase isERK-5, LRKK2, or EphA2.

As inhibitors of protein kinases, the compounds and compositions of thisinvention are particularly useful for treating or lessening the severityof a disease, condition, or disorder where a protein kinase isimplicated in the disease, condition, or disorder. In one aspect, thepresent invention provides a method for treating or lessening theseverity of a disease, condition, or disorder where a protein kinase isimplicated in the disease state. In another aspect, the presentinvention provides a method for treating or lessening the severity of akinase disease, condition, or disorder where inhibition of enzymaticactivity is implicated in the treatment of the disease. In anotheraspect, this invention provides a method for treating or lessening theseverity of a disease, condition, or disorder with compounds thatinhibit enzymatic activity by binding to the protein kinase. Anotheraspect provides a method for treating or lessening the severity of akinase disease, condition, or disorder by inhibiting enzymatic activityof the kinase with a protein kinase inhibitor.

In some embodiments, said method is used to treat or prevent a conditionselected from autoimmune diseases, inflammatory diseases, proliferativeand hyperproliferative diseases, immunologically-mediated diseases, bonediseases, metabolic diseases, neurological and neurodegenerativediseases, cardiovascular diseases, hormone related diseases, allergies,asthma, and Alzheimer's disease. In other embodiments, said condition isselected from a proliferative disorder and a neurodegenerative disorder.

One aspect of this invention provides compounds that are useful for thetreatment of diseases, disorders, and conditions characterized byexcessive or abnormal cell proliferation. Such diseases include, aproliferative or hyperproliferative disease, and a neurodegenerativedisease. Examples of proliferative and hyperproliferative diseasesinclude, without limitation, cancer. The term “cancer” includes, but isnot limited to, the following cancers: breast; ovary; cervix; prostate;testis, genitourinary tract; esophagus; larynx, glioblastoma;neuroblastoma; stomach; skin, keratoacanthoma; lung, epidermoidcarcinoma, large cell carcinoma, small cell carcinoma, lungadenocarcinoma; bone; colon; colorectal; adenoma; pancreas,adenocarcinoma; thyroid, follicular carcinoma, undifferentiatedcarcinoma, papillary carcinoma; seminoma; melanoma; sarcoma; bladdercarcinoma; liver carcinoma and biliary passages; kidney carcinoma;myeloid disorders; lymphoid disorders, Hodgkin's, hairy cells; buccalcavity and pharynx (oral), lip, tongue, mouth, pharynx; small intestine;colon-rectum, large intestine, rectum, brain and central nervous system;chronic myeloid leukemia (CML), and leukemia. The term “cancer”includes, but is not limited to, the following cancers: myeloma,lymphoma, or a cancer selected from gastric, renal, or and the followingcancers: head and neck, oropharangeal, non-small cell lung cancer(NSCLC), endometrial, hepatocarcinoma, Non-Hodgkins lymphoma, andpulmonary.

In some embodiments, the compounds of this invention are useful fortreating cancer, such as colorectal, thyroid, breast, and lung cancer;and myeloproliferative disorders, such as polycythemia vera,thrombocythemia, myeloid metaplasia with myelofibrosis, chronicmyelogenous leukemia, chronic myelomonocytic leukemia, hypereosinophilicsyndrome, juvenile myelomonocytic leukemia, and systemic mast celldisease.

In some embodiments, the compounds of this invention are useful fortreating hematopoietic disorders, in particular, acute-myelogenousleukemia (AMLi), chronic-myelogenous leukemia (CML), acute-promyelocyticleukemia, and acute lymphocytic leukemia (ALL).

Examples of neurodegenerative diseases include, without limitation,Alzheimer's disease.

Another aspect of this invention provides a method for the treatment orlessening the severity of a disease selected from a proliferative orhyperproliterative disease, or a neurodegenerative disease, comprisingadministering an effective amount of a compound, or a pharmaceuticallyacceptable composition comprising a compound, to a subject in needthereof.

The present invention provides compounds, compositions and methods forthe treatment of kinase related disease, particularly MPS1, ERK5, BMK1,MAPK7, polo kinase 1, 2, 3, or 4, Ack1, Ack2, Abl, DCAMKL1, ABL1, Ablmutants, DCAMKL2, ARK5, BRK, MKNK2, FGFR4, TNK1, PLK1, ULK2, PLK4,PRKD1, PRKD2, PRKD3, ROS1, RPS6KA6, TAOK1, TAOK3, TNK2, Bcr-Abl, GAK,cSrc, TPR-Met, Tie2, MET, FGFR3, Aurora, Axl, Bmx, BTK, c-kit, CHK2,Flt3, MST2, p70S6K, PDGFR, PKB, PKC, Raf, ROCK-H, Rsk1, SGK, TrkA, TrkBand TrkC kinase related diseases. In a further embodiment, the kinase isERK-5, LRKK2, or EphA2.

Compounds of the invention modulate the activity of kinases and, assuch, are useful for treating diseases or disorders in which kinases,contribute to the pathology and/or symptomology of the disease. Examplesof kinases that are inhibited by the compounds and compositionsdescribed herein and against which the methods described herein areuseful include, but are not limited to, MPS1, ERK5, BMK1, MAPK7, polokinase 1, 2, 3, or 4, Ack1, Ack2, Abl, DCAMKL1, ABL1, Abl mutants,DCAMKL2, ARK5, BRK, MKNK2, FGFR4, TNK1, PLK1, ULK2, PLK4, PRKD1, PRKD2,PRKD3, ROS1, RPS6KA6, TAOK1, TAOK3, TNK2, Bcr-Abl, GAK, cSrc, TPR-Met,Tie2, MET, FGFR3, Aurora, Axl, Bmx, BTK, c-kit, CHK2, Flt3, MST2,p70S6K, PDGFR, PKB, PKC, Raf, ROCK-H, Rsk1, SGK, TrkA, TrkB and TrkCkinases. In a further embodiment, the kinase is ERK-5, LRKK2, or EphA2.

As inhibitors of protein kinases, the compounds and compositions of thisinvention are also useful in biological samples. One aspect of theinvention relates to inhibiting protein kinase activity in a biologicalsample, which method comprises contacting said biological sample with acompound of the invention or a composition comprising said compound. Theterm “biological sample”, as used herein, means an in vitro or an exvivo sample, including, without limitation, cell cultures or extractsthereof, biopsied material obtained from a mammal or extracts thereof;and blood, saliva, urine, feces, semen, tears, or other body fluids orextracts thereof. Inhibition of protein kinase activity in a biologicalsample is useful for a variety of purposes that are known to one ofskill in the art. Examples of such purposes include, but are not limitedto, blood transfusion, organ-transplantation, and biological specimenstorage.

Another aspect of this invention relates to the study of protein kinasesin biological and pathological phenomena; the study of intracellularsignal transduction pathways mediated by such protein kinases; and thecomparative evaluation of new protein kinase inhibitors. Examples ofsuch uses include, but are not limited to, biological assays such asenzyme assays and cell-based assays.

The activity of the compounds as protein kinase inhibitors may beassayed in vitro, in vivo or in a cell line. In vitro assays includeassays that determine inhibition of either the kinase activity or ATPaseactivity of the activated kinase. Alternate in vitro assays quantitatethe ability of the inhibitor to bind to the protein kinase and may bemeasured either by radiolabelling the inhibitor prior to binding,isolating the inhibitor/kinase complex and determining the amount ofradiolabel bound, or by running a competition experiment where newinhibitors are incubated with the kinase bound to known radioligands.Detailed conditions for assaying a compound utilized in this inventionas an inhibitor of various kinases are set forth in the Examples below.

In accordance with the foregoing, the present invention further providesa method for preventing or treating any of the diseases or disordersdescribed above in a subject in need of such treatment, which methodcomprises administering to said subject a therapeutically effectiveamount of a compound of the invention or a pharmaceutically acceptablesalt thereof. For any of the above uses, the required dosage will varydepending on the mode of administration, the particular condition to betreated and the effect desired.

Pharmaceutical Compositions

In another aspect, the invention provides a pharmaceutical compositioncomprising a compound of formulae I-IX (or formulae A or F), or apharmaceutically acceptable ester, salt, or prodrug thereof, togetherwith a pharmaceutically acceptable carrier.

Compounds of the invention can be administered as pharmaceuticalcompositions by any conventional route, in particular enterally, e.g.,orally, e.g., in the form of tablets or capsules, or parenterally, e.g.,in the form of injectable solutions or suspensions, topically, e.g., inthe form of lotions, gels, ointments or creams, or in a nasal orsuppository form. Pharmaceutical compositions comprising a compound ofthe present invention in free form or in a pharmaceutically acceptablesalt form in association with at least one pharmaceutically acceptablecarrier or diluent can be manufactured in a conventional manner bymixing, granulating or coating methods. For example, oral compositionscan be tablets or gelatin capsules comprising the active ingredienttogether with a) diluents, e.g., lactose, dextrose, sucrose, mannitol,sorbitol, cellulose and/or glycine; b) lubricants, e.g., silica, talcum,stearic acid, its magnesium or calcium salt and/or polyethyleneglycol;for tablets also c) binders, e.g., magnesium aluminum silicate, starchpaste, gelatin, tragacanth, methylcellulose, sodiumcarboxymethylcellulose and or polyvinylpyrrolidone; if desired d)disintegrants, e.g., starches, agar, alginic acid or its sodium salt, oreffervescent mixtures; and/or e) absorbents, colorants, flavors andsweeteners. Injectable compositions can be aqueous isotonic solutions orsuspensions, and suppositories can be prepared from fatty emulsions orsuspensions. The compositions may be sterilized and/or containadjuvants, such as preserving, stabilizing, wetting or emulsifyingagents, solution promoters, salts for regulating the osmotic pressureand/or buffers. In addition, they may also contain other therapeuticallyvaluable substances. Suitable formulations for transdermal applicationsinclude an effective amount of a compound of the present invention witha carrier. A carrier can include absorbable pharmacologically acceptablesolvents to assist passage through the skin of the host. For example,transdermal devices are in the form of a bandage comprising a backingmember, a reservoir containing the compound optionally with carriers,optionally a rate controlling barrier to deliver the compound to theskin of the host at a controlled and predetermined rate over a prolongedperiod of time, and means to secure the device to the skin. Matrixtransdermal formulations may also be used. Suitable formulations fortopical application, e.g., to the skin and eyes, are preferably aqueoussolutions, ointments, creams or gels well-known in the art. Such maycontain solubilizers, stabilizers, tonicity enhancing agents, buffersand preservatives.

Compounds of the invention can be administered in therapeuticallyeffective amounts in combination with one or more therapeutic agents(pharmaceutical combinations). For example, synergistic effects canoccur with other immunomodulatory or anti-inflammatory substances, forexample when used in combination with cyclosporin, rapamycin, orascomycin, or immunosuppressant analogues thereof, for examplecyclosporin A (CsA), cyclosporin G, FK-506, rapamycin, or comparablecompounds, corticosteroids, cyclophosphamide, azathioprine,methotrexate, brequinar, leflunomide, mizoribine, mycophenolic acid,mycophenolate mofetil, 15-deoxyspergualin, immunosuppressant antibodies,especially monoclonal antibodies for leukocyte receptors, for exampleMHC, CD2, CD3, CD4, CD7, CD25, CD28, B7, CD45, CD58 or their ligands, orother immunomodulatory compounds, such as CTLA41g. Where the compoundsof the invention are administered in conjunction with other therapies,dosages of the co-administered compounds will of course vary dependingon the type of co-drug employed, on the specific drug employed, on thecondition being treated and so forth.

The pharmaceutical compositions of the present invention comprise atherapeutically effective amount of a compound of the present inventionformulated together with one or more pharmaceutically acceptablecarriers. As used herein, the term “pharmaceutically acceptable carrier”means a non-toxic, inert solid, semi-solid or liquid filler, diluent,encapsulating material or formulation auxiliary of any type. Thepharmaceutical compositions of this invention can be administered tohumans and other animals orally, rectally, parenterally,intracisternally, intravaginally, intraperitoneally, topically (as bypowders, ointments, or drops), buccally, or as an oral or nasal spray.

Liquid dosage forms for oral administration include pharmaceuticallyacceptable emulsions, microemulsions, solutions, suspensions, syrups andelixirs. In addition to the active compounds, the liquid dosage formsmay contain inert diluents commonly used in the art such as, forexample, water or other solvents, solubilizing agents and emulsifierssuch as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethylacetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyleneglycol, dimethylformamide, oils (in particular, cottonseed, groundnut,corn, germ, olive, castor, and sesame oils), glycerol,tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid estersof sorbitan, and mixtures thereof. Besides inert diluents, the oralcompositions can also include adjuvants such as wetting agents,emulsifying and suspending agents, sweetening, flavoring, and perfumingagents.

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions may be formulated according to the known artusing suitable dispersing or wetting agents and suspending agents. Thesterile injectable preparation may also be a sterile injectablesolution, suspension or emulsion in a nontoxic parenterally acceptablediluent or solvent, for example, as a solution in 1,3-butanediol. Amongthe acceptable vehicles and solvents that may be employed are water,Ringer's solution, U.S.P. and isotonic sodium chloride solution. Inaddition, sterile, fixed oils are conventionally employed as a solventor suspending medium. For this purpose any bland fixed oil can beemployed including synthetic mono- or diglycerides. In addition, fattyacids such as oleic acid are used in the preparation of injectables.

In order to prolong the effect of a drug, it is often desirable to slowthe absorption of the drug from subcutaneous or intramuscular injection.This may be accomplished by the use of a liquid suspension ofcrystalline or amorphous material with poor water solubility. The rateof absorption of the drug then depends upon its rate of dissolutionwhich, in turn, may depend upon crystal size and crystalline form.Alternatively, delayed absorption of a parenterally administered drugform is accomplished by dissolving or suspending the drug in an oilvehicle.

Compositions for rectal or vaginal administration are preferablysuppositories which can be prepared by mixing the compounds of thisinvention with suitable non-irritating excipients or carriers such ascocoa butter, polyethylene glycol or a suppository wax which are solidat ambient temperature but liquid at body temperature and therefore meltin the rectum or vaginal cavity and release the active compound.

Solid compositions of a similar type may also be employed as fillers insoft and hard-filled gelatin capsules using such excipients as lactoseor milk sugar as well as high molecular weight polyethylene glycols andthe like.

The active compounds can also be in micro-encapsulated form with one ormore excipients as noted above. The solid dosage forms of tablets,dragees, capsules, pills, and granules can be prepared with coatings andshells such as enteric coatings, release controlling coatings and othercoatings well known in the pharmaceutical formulating art. In such soliddosage forms the active compound may be admixed with at least one inertdiluent such as sucrose, lactose or starch. Such dosage forms may alsocomprise, as is normal practice, additional substances other than inertdiluents, e.g., tableting lubricants and other tableting aids such amagnesium stearate and microcrystalline cellulose. In the case ofcapsules, tablets and pills, the dosage forms may also comprisebuffering agents.

Dosage forms for topical or transdermal administration of a compound ofthis invention include ointments, pastes, creams, lotions, gels,powders, solutions, sprays, inhalants or patches. The active componentis admixed under sterile conditions with a pharmaceutically acceptablecarrier and any needed preservatives or buffers as may be required.Ophthalmic formulation, ear drops, eye ointments, powders and solutionsare also contemplated as being within the scope of this invention.

The ointments, pastes, creams and gels may contain, in addition to anactive compound of this invention, excipients such as animal andvegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulosederivatives, polyethylene glycols, silicones, bentonites, silicic acid,talc and zinc oxide, or mixtures thereof.

Powders and sprays can contain, in addition to the compounds of thisinvention, excipients such as lactose, talc, silicic acid, aluminumhydroxide, calcium silicates and polyamide powder, or mixtures of thesesubstances. Sprays can additionally contain customary propellants suchas chlorofluorohydrocarbons.

Transdermal patches have the added advantage of providing controlleddelivery of a compound to the body. Such dosage forms can be made bydissolving or dispensing the compound in the proper medium. Absorptionenhancers can also be used to increase the flux of the compound acrossthe skin. The rate can be controlled by either providing a ratecontrolling membrane or by dispersing the compound in a polymer matrixor gel.

According to the methods of treatment of the present invention,disorders are treated or prevented in a subject, such as a human orother animal, by administering to the subject a therapeuticallyeffective amount of a compound of the invention, in such amounts and forsuch time as is necessary to achieve the desired result. The term“therapeutically effective amount” of a compound of the invention, asused herein, means a sufficient amount of the compound so as to decreasethe symptoms of a disorder in a subject. As is well understood in themedical arts a therapeutically effective amount of a compound of thisinvention will be at a reasonable benefit/risk ratio applicable to anymedical treatment.

In general, compounds of the invention will be administered intherapeutically effective amounts via any of the usual and acceptablemodes known in the art, either singly or in combination with one or moretherapeutic agents. A therapeutically effective amount may vary widelydepending on the severity of the disease, the age and relative health ofthe subject, the potency of the compound used and other factors. Ingeneral, satisfactory results are indicated to be obtained systemicallyat daily dosages of from about 0.03 to 2.5 mg/kg per body weight. Anindicated daily dosage in the larger mammal, e.g. humans, is in therange from about 0.5 mg to about 100 mg, conveniently administered, e.g.in divided doses up to four times a day or in retard form. Suitable unitdosage forms for oral administration comprise from ca. 1 to 50 mg activeingredient.

In certain embodiments, a therapeutic amount or dose of the compounds ofthe present invention may range from about 0.1 mg/Kg to about 500 mg/Kg,alternatively from about 1 to about 50 mg/Kg. In general, treatmentregimens according to the present invention comprise administration to apatient in need of such treatment from about 10 mg to about 1000 mg ofthe compound(s) of this invention per day in single or multiple doses.Therapeutic amounts or doses will also vary depending on route ofadministration, as well as the possibility of co-usage with otheragents.

Upon improvement of a subject's condition, a maintenance dose of acompound, composition or combination of this invention may beadministered, if necessary. Subsequently, the dosage or frequency ofadministration, or both, may be reduced, as a function of the symptoms,to a level at which the improved condition is retained when the symptomshave been alleviated to the desired level, treatment should cease. Thesubject may, however, require intermittent treatment on a long-termbasis upon any recurrence of disease symptoms.

It will be understood, however, that the total daily usage of thecompounds and compositions of the present invention will be decided bythe attending physician within the scope of sound medical judgment. Thespecific inhibitory dose for any particular patient will depend upon avariety of factors including the disorder being treated and the severityof the disorder; the activity of the specific compound employed; thespecific composition employed; the age, body weight, general health, sexand diet of the patient; the time of administration, route ofadministration, and rate of excretion of the specific compound employed;the duration of the treatment; drugs used in combination or coincidentalwith the specific compound employed; and like factors well known in themedical arts.

The invention also provides for a pharmaceutical combinations, e.g. akit, comprising a) a first agent which is a compound of the invention asdisclosed herein, in free form or in pharmaceutically acceptable saltform, and b) at least one co-agent. The kit can comprise instructionsfor its administration.

The terms “co-administration” or “combined administration” or the likeas utilized herein are meant to encompass administration of the selectedtherapeutic agents to a single patient, and are intended to includetreatment regimens in which the agents are not necessarily administeredby the same route of administration or at the same time.

The term “pharmaceutical combination” as used herein means a productthat results from the mixing or combining of more than one activeingredient and includes both fixed and non-fixed combinations of theactive ingredients. The term “fixed combination” means that the activeingredients, e.g. a compound of the invention and a co-agent, are bothadministered to a patient simultaneously in the form of a single entityor dosage. The term “non-fixed combination” means that the activeingredients, e.g. a compound of the invention and a co-agent, are bothadministered to a patient as separate entities either simultaneously,concurrently or sequentially with no specific time limits, wherein suchadministration provides therapeutically effective levels of the twocompounds in the body of the patient. The latter also applies tococktail therapy, e.g. the administration of three or more activeingredients.

In certain embodiments, these compositions optionally further compriseone or more additional therapeutic agents. For example, chemotherapeuticagents or other antiproliferative agents may be combined with thecompounds of this invention to treat proliferative diseases and cancer.Examples of known chemotherapeutic agents include, but are not limitedto, Gleevec™, adriamycin, dexamethasone, vincristine, cyclophosphamide,fluorouracil, topotecan, taxol, interferons, and platinum derivatives.

Other examples of agents the compounds of this invention may also becombined with include, without limitation: treatments for Alzheimer'sDisease such as Aricept18 and Excelon®; treatments for Parkinson'sDisease such as L-DOPA/carbidopa, entacapone, ropinrole, pramipexole,bromocriptine, pergolide, trihexephendyl, and amantadine; agents fortreating Multiple Sclerosis (MS) such as beta interferon (e.g., Avonex®and Rebif®), Copaxone®, and mitoxantrone; treatments for asthma such asalbuterol and Singulair®; agents for treating schizophrenia such aszyprexa, risperdal, seroquel, and haloperidol; anti-inflammatory agentssuch as corticosteroids, TNF blockers, IL-I RA, azathioprine,cyclophosphamide, and sulfasalazine; immunomodulatory andimmunosuppressive agents such as cyclosporin, tacrolimus, rapamycin,mycophenolate mofetil, interferons, corticosteroids, cyclophophamide,azathioprine, and sulfasalazine; neurotrophic factors such asacetylcholinesterase inhibitors, MAO inhibitors, interferons,anti-convulsants, ion channel blockers, riluzole, and antiparkinsonianagents; agents for treating cardiovascular disease such asbeta-blockers, ACE inhibitors, diuretics, nitrates, calcium channelblockers, and statins; agents for treating liver disease such ascorticosteroids, cholestyramine, interferons, and antiviral agents;agents for treating blood disorders such as corticosteroids,antileukemic agents, and growth factors; and agents for treatingimmunodeficiency disorders such as gamma globulin. Some examples ofmaterials which can serve as pharmaceutically acceptable carriersinclude, but are not limited to, ion exchangers, alumina, aluminumstearate, lecithin, serum proteins, such as human serum albumin, buffersubstances such as phosphates, glycine, sorbic acid, or potassiumsorbate, partial glyceride mixtures of saturated vegetable fatty acids,water, salts or electrolytes, such as protamine sulfate, disodiumhydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zincsalts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone,polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, woolfat, sugars such as lactose, glucose and sucrose; starches such as cornstarch and potato starch; cellulose and its derivatives such as sodiumcarboxymethyl cellulose, ethyl cellulose and cellulose acetate; powderedtragacanth; malt; gelatin; talc; excipients such as cocoa butter andsuppository waxes, oils such as peanut oil, cottonseed oil; saffloweroil; sesame oil; olive oil; corn oil and soybean oil; glycols; such apropylene glycol or polyethylene glycol; esters such as ethyl oleate andethyl laurate, agar; buffering agents such as magnesium hydroxide andaluminum hydroxide; alginic acid; pyrogen-free water, isotonic saline;Ringer's solution; ethyl alcohol, and phosphate buffer solutions, aswell as other non-toxic compatible lubricants such as sodium laurylsulfate and magnesium stearate, as well as coloring agents, releasingagents, coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the composition,according to the judgment of the formulator. The protein kinaseinhibitors or pharmaceutical salts thereof may be formulated intopharmaceutical compositions for administration to animals or humans.These pharmaceutical compositions, which comprise an amount of theprotein inhibitor effective to treat or prevent a proteinkinase-mediated condition and a pharmaceutically acceptable carrier, areanother embodiment of the present invention.

In another aspect, the invention provides a kit comprising a compoundcapable of inhibiting kinase activity selected from one or morecompounds of formulae I-IX (or formulae A or F), and instructions foruse in treating cancer.

EXAMPLES

The compounds and processes of the present invention will be betterunderstood in connection with the following examples, which are intendedas an illustration only and not to limit the scope of the invention.Various changes and modifications to the disclosed embodiments will beapparent to those skilled in the art and such changes and modificationsincluding, without limitation, those relating to the chemicalstructures, substituents, derivatives, formulations and/or methods ofthe invention may be made without departing from the spirit of theinvention and the scope of the appended claims.

The following synthesis examples illustrate suitable methods forpreparing compounds of the invention.

Example 1: Synthesis of Thiophene Compounds of Formula IV

To a stirred solution of methyl 3-aminothiophene-2-carboxylate (2.36 g,15 mmol) and DIEA (5.22 mL, 30 mmol) in 2-PrOH (60 mL) was added2,4-dichloro-5-nitropyrimidine (3.78 g, 19.5 mmol) in one portion atroom temperature. Then the reaction was stirred at RT. After thereaction complete as monitored by LC-MS, the resulting mixture wasdiluted with ethyl acetate and washed with water and brine, the organiclayer was dried over anhydrous sodium sulfate and concentrated in vacuo.The residue was used for next step directly without furtherpurification.

A mixture of methyl3-(2-chloro-5-nitropyrimidin-4-ylamino)thiophene-2-carboxylate and ironpower (8.4 g, 150 mmol) in acetic acid (220 mL) was heated at 55° C.After the reaction complete, the mixture was concentrated in vacuo. Thenthe residue was purified by silica-gel column chromatography withmethanol and dichloromethane to give methyl3-(5-amino-2-chloropyrimidin-4-ylamino)thiophene-2-carboxylate (4.2 g,98%).

To a suspension of methyl3-(5-amino-2-chloropyrimidin-4-ylamino)thiophene-2-carboxylatein inmethanol/tetrahydrofuran (30 mL/30 mL) was added LiOH solution (3.99 g(95 mmol) in 30 mL water) at room temperature. After the reactioncomplete as monitored by LC-MS, the reaction mixture was concentratedand neutralized with 6 N HCl till PH to 5. The precipitated solid wascollected. The aqueous layer was extracted with CHCl3/i-PrOH (4/1)twice. The organic layer was washed with brine and dried over anhydroussodium sulfate. The solvent was removed in vacuo. This portion ofproduct was combined with the precipitated solid.

A reaction mixture of3-(5-amino-2-chloropyrimidin-4-ylamino)thiophene-2-carboxylic acid (1.17g, 4.33 mmol), HATU (2.47 g, 6.5 mmol) and DIEA (2.27 mL, 13 mmol) in25.0 mL of dimethyl sulfoxide (DMSO) was stirred at room temperature.After the reaction complete as monitored by LC-MS, the solution waspoured into ice-water. The precipitated solid was collected andlyophilyzed. The crude product was used in next step without furtherpurification.

A mixture of starting material (25 mg, 0.1 mmol),2-methoxy-4-(4-methylpiperazin-1-yl)benzenamine (22 mg, 0.1 mmol),X-Phos (4.3 mg), Pd₂(dba)₃ (5.5 mg) and K₂CO₃ (41.5 mg, 0.3 mmol) int-BuOH (1.5 mL) was heated at 100° C. in a seal tube for 4 h. Then thereaction was filtered through celite and eluted with dichloromethane.The solvent was removed in vacuo and the residue was purified by ISCO toafford the title compound (24.7 mg).

Example 2: Synthesis of Sulfone Compounds of Formula III

To a stirred solution of 2,4-dichloropyrimidin-5-amine (328 mg, 2.0mmol) and TEA (0.42 mL, 3.0 mmol) in dichloromethane (6.0 mL) was added2-nitrobenzene-1-sulfonyl chloride (443 mg, 2.0 mmol) in one portion at0° C. The reaction mixture was stirred and allowed to approach roomtemperature. After the reaction complete as monitored by LC-MS, theresulting mixture was diluted with dichloromethane and washed with waterand brine, the organic layer was dried over anhydrous sodium sulfate andconcentrated in vacuo. The residue was purified by silica gelchromatography with dichloromethane:methanol to give the desiredcompound N-(2,4-dichloropyrimidin-5-yl)-2-nitrobenzenesulfonamide (359mg, 52%).

A mixture of N-(2,4-dichloropyrimidin-5-yl)-2-nitrobenzenesulfonamide(190 mg, 0.55 mmol) and iron power (560 mg, 10 mmol) in acetic acid (8mL) was heated at 60° C. After the reaction complete, the mixture wasconcentrated in vacuo and poured into ice-water. The precipitated solidwas collected and lyophilyzed. The crude product was used in next stepwithout further purification.

A mixture of starting material (28 mg, 0.1 mmol),2-methoxy-4-(4-methylpiperazin-1-yl)benzenamine (22 mg, 0.1 mmol),X-Phos (4.3 mg), Pd₂(dba)₃ (5.5 mg) and K₂CO₃ (41.5 mg, 0.3 mmol) int-BuOH (1.5 mL) was heated at 100° C. in a seal tube for 4 h. Thereaction was then filtered through celite, eluted with dichloromethane,and concentrated in vacuo. The residue was then purified byreverse-phase prep-HPLC to afford the title compound as the TFA salt(7.2 mg, 15%).

Example 3: Synthesis of Tetracyclic Compounds of Formula I

A mixture of 1-(tert-butoxycarbonyl)indoline-7-carboxylic acid (460 mg,1.75 mmol), MeI (0.22 mL, 3.5 mmol) and potassium carbonate (484 mg, 3.5mmol) in acetone (20 mL) was heated at 60° C. After the reaction wascomplete, the reaction mixture was filtered through celite, eluted withdichloromethane and concentrated in vacuo. The residue was purified bysilica gel chromatography with hexanes:ethyl acetate to give1-tert-butyl 7-methyl indoline-1,7-dicarboxylate (390 mg, 80%).

A mixture of 1-tert-butyl 7-methyl indoline-1,7-dicarboxylate (390 mg,1.41 mmol) and TFA (2.5 mL) in dichloromethane (8 mL) was stirred atroom temperature. After the reaction complete as monitored by LC-MS, themixture was concentrated in vacuo. The residue was dissolved in ethylacetate and washed with saturated sodium bicarbonate. The organic layerwas dried over anhydrous sodium sulfate and concentrated in vacuo. Thecrude product was used in next step without further purification.

A mixture of methyl 1-indoline-7-carboxylate (244 mg, 1.37 mmol),2,4-dichloro-5-nitropyrimidine (400 mg, 2.06 mmol) and DIEA (0.72 mL,4.1 mmol) in dioxane (17 mL) was stirred at 50° C. After the reactioncomplete as monitored by LC-MS, the resulting mixture was concentratedin vacuo and purified by silica gel chromatography to give methyl1-(2-chloro-5-nitropyrimidin-4-yl)indoline-7-carboxylate (433 mg, 94%).

A mixture of methyl1-(2-chloro-5-nitropyrimidin-4-yl)indoline-7-carboxylate (188 mg, 0.69mmol) and iron power (800 mg, 14.3 mmol) in acetic acid (12 mL) washeated at 55° C. After the reaction complete, the mixture wasconcentrated in vacuo and poured into ice-water. The precipitated solidwas collected and lyophilyzed. The crude product was used in next stepwithout further purification.

A mixture of starting material (27 mg, 0.1 mmol),2-methoxy-4-(4-methylpiperazin-1-yl)benzenamine (22 mg, 0.1 mmol),X-Phos (4.3 mg), Pd₂(dba)₃ (5.5 mg) and K₂CO₃ (41.5 mg, 0.3 mmol) int-BuOH (1.5 mL) was heated at 100° C. in a seal tube for 4 h. Thereaction was then filtered through celite, eluted with dichloromethane,and concentrated in vacuo. The residue was then purified byreverse-phase prep-HPLC to afford the title compound as the TFA salt(21.5 mg, 47%).

Example 4: Synthesis of Compounds of Formula II

A mixture of 2,4-dichloropyrimidin-5-amine (246 mg, 1.5 mmol),2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzenamine (219 mg, 1.0mmol), Pd(dppb)Cl₂ (60 mg, 0.1 mmol) and Na₂CO₃ (4 mL, 1.0 M solution)in toluene/EtOH (2.0 mL/3.0 mL) was heated at 100° C. in a seal tube for3 h. The reaction was then filtered through celite, eluted withdichloromethane, washed by brine, and concentrated in vacuo. The residuewas then purified by silica gel chromatography with 3.5 N ammonia inMeOH solution: dichloromethane to give4-(2-aminophenyl)-2-chloropyrimidin-5-amine (158 mg, 48%).

To a stirred solution of 4-(2-aminophenyl)-2-chloropyrimidin-5-amine (17mg, 0.077 mmol) in dioxane (2.5 mL) were added DIEA (0.04 mL) andtriphosgene (11 mg, 0.039 mmol) at room temperature. Then the reactionwas stirred at 100° C. After the reaction complete as monitored byLC-MS, the solution was poured into ice-water. The precipitated solidwas collected and lyophilyzed. The crude product was used in next stepwithout further purification.

A mixture of starting material (12 mg, 0.05 mmol),1-(4-aminophenyl)piperidin-4-ol (10 mg, 0.05 mmol), X-Phos (4.3 mg),Pd₂(dba)₃ (5.5 mg) and K₂CO₃ (30 mg, 0.22 mmol) in t-BuOH (1.0 mL) washeated at 85° C. in a seal tube for 4 h. The reaction was then filteredthrough celite, eluted with dichloromethane, and concentrated in vacuo.The residue was then purified by reverse-phase prep-HPLC to afford thetitle compound as the TFA salt (8.2 mg, 40%).

Example 5: Synthesis of Compounds of Formula V

Chemistry.

An efficient four-step synthetic route was developed to enable thesynthesis of benzo[e]pyrimido-[5,4-b]diazepine-6(11H)-ones. Thesynthesis of 25 using a modified synthetic procedure is outlined inScheme 5. First, 2,4-dichloro-5-nitropyrimidine was reacted withN-cyclopentylanthranilic ethyl ester under acidic conditions using 4 Nof hydrochloride in dioxane at 60° C. to give the amination product 12in good yield. Using basic conditions with diisopropylethyl amine, onlytrace quantities of the amination product 12 were obtained and2,4-dichloro-5-nitropyrimidine was hydrolyzed completely. We suspectthat the substituted reaction under basic condition was hampered by thesteric hindrance imposed by the bulky N-cyclopentylanthranilic ethylester substrate. We also observed that the yield of the substitutionreaction under acidic conditions is higher than that obtained underbasic condition when the N-isopropyl anthranilic ethyl ester is used asa substrate. The substitution reaction was followed by iron-mediatedreduction of 12 and in situ cyclization in acetic acid at 60° C. toafford the 7-member lactam intermediate 13 in good yield. Compound 25was obtained via methylation of 13 of the lactam followed by palladiummediated amination of 14 with(4-amino-3-methoxyphenyl)(4-(4-methylpiperazin-1-yl)piperidin-1-yl)methanone.

In more detail:

A mixture of ethyl 2-(cyclopentylamino)benzoate (1.40 g, 6.0 mmol), 4 NHCl in dioxane solution (2.25 mL, 9.0 mmol) and2,4-dichloro-5-nitropyrimidine (1.74 g, 9.0 mmol) in dioxane (40 mL) washeated at 60° C. for 90 hours. After the reaction was complete asmonitored by thin layer chromatography (TLC), the reaction solution wasconcentrated and the residue was purified by silica-gel columnchromatography with ethyl acetate and hexane (1/20, v/v) to give theamination product 12 (1.84 g, 79%). MS (ESI) m/z 391 (M+H)⁺.

A mixture of compound 12 (1.79 g, 4.59 mmol) and iron powder (2.57 g,45.9 mmol) in acetic acid (80 mL) was heated at 60° C. for 9 hours.After the reaction was complete as monitored by reverse phase analyticalliquid-chromatography electrospray mass spectrometry (LC-MS), thesolvent was removed in vacuo. The resulting residue was poured intoice-water which resulted in a solid precipitate that was collected byfiltration, washed with water and air dried to give the intermediate 13(1.21 g, 84%). ¹H NMR (600 MHz, DMSO-d₆) δ 10.48 (s, 1H), 8.18 (s, 1H),7.57 (d, J=7.2 Hz, 1H), 7.50 (s, 1H), 7.30 (d, J=7.2 Hz, 1H), 7.17 (s,1H), 4.61 (brs, 1H), 2.30-1.90 (m, 2H), 1.70-1.40 (m, 4H), 1.38-1.20 (m,2H). MS (ESI) m/z 315 (M+H)⁺.

To a stirred suspension of compound 13 (314 mg, 1.0 mmol) and MeI (0.13mL, 2.0 mmol) in dimethyl acetamide (DMA, 10.0 mL) was added NaH (80 mg,60% suspension in mineral oil) at −10° C. and the reaction was graduallywarmed to 0° C. After the reaction was complete as monitored by LC-MS,the solution was poured into ice-water which resulted in a solidprecipitate. The precipitate was collected by filtration, washed withwater and air dried to give the intermediate 14 (273 mg, 83%). ¹H NMR(600 MHz, DMSO-d₆) δ 8.64 (s, 1H), 7.58 (dd, J=1.8, 7.8 Hz, 1H), 7.49(t, J=7.8 Hz, 1H), 7.33 (d, J=8.4 Hz, 1H), 7.19 (t, J=7.2 Hz, 1H),4.68-4.64 (m, 1H), 3.44 (s, 3H), 2.28-2.20 (m, 1H), 2.10-2.02 (m, 1H),1.64-1.54 (m, 4H), 1.50-1.34 (m, 2H). MS (ESI) m/z 329 (M+H)⁺.

A mixture of 14 (33 mg, 0.1 mmol),(4-amino-3-methoxyphenyl)(4-(4-methylpiperazin-1-yl)piperidin-1-yl)methanone(33 mg, 0.1 mmol), X-Phos (4.3 mg), Pd₂(dba)₃ (5.5 mg) and K₂CO₃ (41.5mg, 0.3 mmol) in 1.2 mL of t-BuOH was heated at 100° C. in a seal tubefor 4 h. Then the reaction was filtered through Celite and eluted withdichloromethane. The dichloromethane was removed in vacuo and theresulting crude product was purified by reverse-phase prep-HPLC using awater (0.05% TFA)/acetonitrile (0.05% TFA) gradient to afford the titlecompound 25 (35.3 mg, 57%). ¹H NMR (600 MHz, DMSO-d₆) δ 9.53 (s, 1H),8.44 (s, 1H), 8.20 (d, J=7.8 Hz, 1H), 8.14 (s, 1H), 7.56 (d, J=8.4 Hz,1H), 7.45 (t, J=7.8 Hz, 1H), 7.28 (d, J=9.0 Hz, 1H), 7.15 (t, J=7.8 Hz,1H), 6.99 (d, J=8.4 Hz, 1H), 4.68-4.64 (m, 1H), 3.86 (s, 3H), 3.42 (s,3H), 3.40-3.28 (m, 6H), 3.08-2.90 (m, 5H), 2.76 (s, 3H), 2.60-2.52 (m,1H), 2.46-2.38 (m, 1H), 2.30-2.27 (m, 1H), 2.09-2.06 (m, 1H), 1.85-1.65(m, 2H), 1.57-1.52 (m, 4H), 1.50-1.47 (m, 1H), 1.42-1.32 (m, 3H). MS(ESI) m/z 625 (M+H)⁺.

Example 6: Synthesis of Compounds of Formula VII

To a stirred solution of 2,4-dichloro-5H-pyrrolo[3,2-d]pyrimidine (1.01g, 5.4 mmol) in DMF (40 mL) was added sodium hydride (0.43 g, 10.8 mmol)at 0° C. After 15 minutes, 2-nitrobenzoyl chloride (0.864 mL, 6.5 mmol)was added dropwise. The reaction was stirred at 0° C. till the reactioncompleted as monitored by LC-MS. The reaction was quenched by saturatedNH₄Cl solution, poured into ice-water. The solid was collected, driedunder vacuum and used for next step directly without furtherpurification.

A mixture of(2,4-dichloro-5H-pyrrolo[3,2-d]pyrimidin-5-yl)(2-nitrophenyl)methanone(1.04 g, 3.1 mmol) and iron power (1.8 g, 32.1 mmol) in acetic acid (50mL) was heated at 55° C. After the reaction complete, the mixture wasconcentrated in vacuo and poured into ice-water. The precipitated solidwas collected and lyophilyzed. The crude product was used in next stepwithout further purification.

To a stirred suspension of4-chloro-3,5,6,11a-tetraazadibenzo[cd,g]azulen-11(6H)-one (270 mg, 1mmol) in DMA (8 mL), Met (0.094 mL, 1.5 mmol) was added. The reactionwas stirred at 0° C. till the reaction completed as monitored by LC-MS.Then the reaction was quenched by saturated NH₄Cl solution, poured intoice-water. The precipitated solid was collected. The aqueous layer wasextracted with ethyl acetate. The organic layer was washed with brineand dried over anhydrous sodium sulfate. The solvent was removed invacuo. This portion of product was combined with the precipitated solidand was purified by silica gel chromatography to give the desiredcompound (210 mg, 74%).

A mixture of starting material (29 mg, 0.1 mmol),2-methoxy-4-(4-methylpiperazin-1-yl)benzenamine (22 mg, 0.1 mmol),X-Phos (4.3 mg), Pd₂(dba)₃ (5.5 mg) and K₂CO₃ (41.5 mg, 0.3 mmol) int-BuOH (1.5 mL) was heated at 85° C. in a seal tube for 1.5 h. Then thereaction was filtered through celite and eluted with dichloromethane.The solvent was removed in vacuo and the residue was purified by ISCO toafford the title compound (8.1 mg).

Example 7: Synthesis of Compounds of Formula VIII

A reaction mixture of 2,4-dichloro-5-nitropyrimidine (2.91 g, 65 mmol),ethyl 2-hydroxybenzoate (1.66 g, 10 mmol) and DIEA (3.5 mL, 20 mmol) indichloromethane (45 mL) was stirred at 0° C. till the reaction completedas monitored by LC-MS. The reaction was diluted with ethyl acetate andwashed with water and brine, the organic layer was dried over anhydroussodium sulfate, concentrated in vacuo and used for next step directlywithout further purification.

A mixture of ethyl 2-((2-chloro-5-nitropyrimidin-4-yl)oxy)benzoate (3.2g, 10 mmol) and iron power (5.6 g, 100 mmol) in acetic acid (140 mL) washeated at 60° C. After the reaction complete, the mixture wasconcentrated in vacuo and poured into ice-water. The precipitated solidwas collected and lyophilyzed. The crude product was used in next stepwithout further purification.

To a stirred solution of ethyl2-((5-amino-2-chloropyrimidin-4-yl)oxy)benzoate (1.47 g, 5.0 mmol) inCH₂Cl₂ (50 mL) was added Al(CH₃)₃ solution (0.15 mmol) at 0° C. Thereaction mixture was heated to 45° C. slowly. When the reactioncompleted as monitored by LC-MS, it was cooled to 0° C. and quenched byaddition of 1N HCl. The mixture was concentrated in vacuo and pouredinto ice-water. The precipitated solid was collected and lyophilyzed.The crude product was used in next step without further purification.

To a stirred suspension of2-chlorobenzo[f]pyrimido[4,5-b][1,4]oxazepin-6(5H)-one (247 mg, 1 mmol)in DMA (10 mL), Met (0.08 mL, 1.5 mmol) was added. The reaction wasstirred at −10° C. till the reaction completed as monitored by LC-MS.Then the reaction was quenched by saturated NH₄Cl solution, poured intoice-water. The precipitated solid was collected. The aqueous layer wasextracted with ethyl acetate. The organic layer was washed with brineand dried over anhydrous sodium sulfate. The solvent was removed invacuo. This portion of product was combined with the precipitated solidand was purified by silica gel chromatography to give the desiredcompound.

A mixture of starting material (29 mg, 0.1 mmol),4-((4-ethylpiperazin-1-yl)methyl)-3-(trifluoromethyl)aniline (29 mg, 0.1mmol), X-Phos (4.3 mg), Pd₂(dba)₃ (5.5 mg) and K₂CO₃ (41.5 mg, 0.3 mmol)in t-BuOH (1.5 mL) was heated at 100° C. in a seal tube for 4 h. Thenthe reaction was filtered through celite and eluted withdichloromethane. The solvent was removed in vacuo and the residue waspurified by HPLC to afford the title compound (26.3 mg).

Example 8: Synthesis of Additional Compounds

Methyl 3-amino-2-naphthoate

To a solution of 3-amino-2-naphthoic acid (562 mg, 3.0 mmol, 1.0 eq) inmethanol/toluene (1:4, 10 mL) was added 2.0 M of TMSCH₂N₂ solution inhexane (1.8 mL, 3.6 mmol, 1.2 eq) at 0° C. The reaction was stirredovernight at rt. Next day, the reaction was quenched with excess aceticacid until no bubbling was seen. The mixture was directly concentratedin vacuo. The residue was purified by silica-gel column chromatographywith ethyl acetate and hexane (0%-25% gradient, v/v) to give compound 1(500 mg, 83%). ¹H NMR (400 MHz, MeOD) δ 8.46 (s, 1H), 7.70 (d, J=8.2 Hz,1H), 7.51 (d, J=8.2 Hz, 1H), 7.37 (ddd, J=8.2, 6.8, 1.2 Hz, 1H), 7.15(ddd, J=8.2, 6.8, 1.2 Hz, 1H), 7.05 (s, 1H), 3.93 (s, 3H). MS (ESI)calcd for [C₁₂H₁₂NO₂]⁺: 202; found 202.

Methyl 3-((2-chloro-6-methyl-5-nitropyrimidin-4-yl)amino)-2-naphthoate

A mixture of compound 1 (480 mg, 2.4 mmol, 1.0 eq),N,N-diisopropylethylamine (DIEA) (0.83 mL, 4.8 mmol, 2.0 eq) and2,4-dichloro-6-methyl-5-nitropyrimidine (0.76 g, 3.6 mmol, 1.5 eq) in2-propanol (43 mL) was stirred at rt overnight. The product crashed outof 2-propanol, and was collected by filtration and dried in vacuo. Thecrude compound 2 (0.79 g, 88%) was used for the next step withoutfurther purification. ¹H NMR (400 MHz, CDCl₃) δ 12.04 (s, 1H), 8.94 (s,1H), 8.68 (s, 1H), 7.89 (d, J=8.2 Hz, 2H), 7.63 (ddd, J=8.2, 7.0, 1.2Hz, 1H), 7.51 (ddd, J=8.2, 7.0, 1.2 Hz, 1H), 4.05 (s, 3H), 2.73 (s, 3H).MS (ESI) calcd for [C₁₇H₁₄ClN₄O₄]⁺: 373; found 373.

2-chloro-4-methyl-5,13-dihydro-6H-naphtho[2,3-e]pyrimido[5,4-b][1,4]diazepin-6-one

To a solution of compound 2 (0.79 g, 2.1 mmol, 1.0 eq) in acetic acid(90 mL) was added iron powder (1.7 g, 30.4 mmol, 14.5 eq). The reactionwas stirred at 60° C. overnight. After the reaction was complete asmonitored by reverse phase analytical liquid-chromatography electrospraymass spectrometry (LC-MS), the solvent was removed in vacuo. Theresulting residue was poured into ice water and stirred, which resultedin a solid precipitate that was collected by filtration, washed withwater and air dried to give compound 3 (0.64 g, 98%). ¹H NMR (400 MHz,CDCl₃) δ 8.63 (s, 1H), 7.85 (d, J=8.2 Hz, 1H), 7.68 (d, J=8.2 Hz, 1H),7.54 (ddd, J=8.2, 7.0, 1.4 Hz, 1H), 7.42 (ddd, J=8.2, 7.0, 1.2 Hz, 1H),7.17 (s, 1H), 7.15 (s, 1H), 6.88 (s, 1H), 2.52 (s, 3H). MS (ESI) calcdfor [C₁₆H₁₂ClN₄O]⁺: 311; found 311.

2-chloro-4,5,13-trimethyl-5,13-dihydro-6H-naphtho[2,3-e]pyrimido[5,4-b][1,4]diazepin-6-one

To a stirred suspension of compound 3 (0.64 g, 2.1 mmol, 1.0 eq) and Met(0.64 mL, 10.3 mmol, 5.0 eq) in dimethyl acetamide (DMA, 20.0 mL) wasadded NaH (300 mg, 60% suspension in mineral oil, 3.6 eq) at 0° C. Afterthe reaction was complete as monitored by LC-MS, the solution was pouredinto ice water, which resulted in a solid precipitate. The precipitatewas collected by filtration, washed with water and air dried to give thecrude product. The crude product was purified by silica-gel columnchromatography with ethyl acetate and hexane (0%-80% gradient, v/v) togive compound 4 (67 mg, 10%). ¹H NMR (400 MHz, MeOD) δ 8.30 (s, 1H),7.88 (d, J=8.2 Hz, 1H), 7.84 (d, J=8.2 Hz, 1H), 7.61 (s, 1H), 7.54 (ddd,J=8.2, 7.0, 1.2 Hz, 1H), 7.45 (ddd, J=8.2, 7.0, 1.2 Hz, 1H), 3.51 (s,3H), 3.37 (s, 3H), 2.48 (s, 3H). MS (ESI) calcd for [C₁₈H₁₆ClN₄O]⁺: 339;found 339.

4,5,13-trimethyl-2-((1-(piperidin-4-yl)-1H-pyrazol-4-yl)amino)-5,13-dihydro-6H-naphtho[2,3-e]pyrimido[5,4-b][1,4]diazepin-6-one

A mixture of 4 (34 mg, 0.1 mmol, 1.0 eq), tert-butyl4-(4-amino-1H-pyrazol-1-yl)piperidine-1-carboxylate (27 mg, 0.1 mmol,1.0 eq), X-Phos (8.6 mg, 20%), Pd₂(dba)₃ (11 mg, 10%) and K₂CO₃ (41.5mg, 0.3 mmol) in 1.2 mL of t-BuOH was heated at 85° C. in a sealed tubefor 3.5 h. The reaction was then filtered through celite and eluted withdichloromethane (DCM). The DCM was removed in vacuo. The resulting crudeproduct was stirred with trifluoroacetic acid (TFA, 0.38 mL, 5 mmol, 50eq) in DCM (2 mL) at rt overnight to afford Boc deprotection. Thesolvent was removed in vacuo. The residue was purified by reverse-phaseprep-HPLC using a water (0.05% TFA)/methanol (0.05% TFA) gradient toafford the title compound HTH-01-015 as TFA salt (18 mg, yield: 31%). ¹HNMR (400 MHz, DMSO-d₆) δ 9.72-9.40 (br, 1H), 8.74-8.61 (br, 1H),8.54-8.37 (br, 1H), 8.29 (s, 1H), 7.97 (d, J=8.2 Hz, 1H), 7.92 (s, 1H),7.88 (d, J=8.2 Hz, 1H), 7.67 (s, 1H), 7.60 (s, 1H), 7.56 (ddd, J=8.2,7.0, 1.2 Hz, 1H), 7.46 (ddd, J=8.2, 7.0, 1.2 Hz, 1H), 4.54-4.41 (br,1H), 3.54-3.38 (br, 5H), 3.27 (s, 3H), 3.17-3.02 (br, 2H), 2.33 (s, 3H),2.26-2.04 (br, 4H). MS (ESI) calcd for [C₂₆H₂₉N₈O⁺]⁺: 469; found 469.

XMD18-42 and XMD17-51 were synthesized following similar strategies asshown in Scheme 8.

5,13-dimethyl-2-((1-(piperidin-4-yl)-1H-pyrazol-4-yl)amino)-5,13-dihydro-6H-naphtho[2,3-e]pyrimido[5,4-b][1,4]diazepin-6-one

¹H NMR (400 MHz, DMSO-d₆) δ 9.77-9.44 (br, 1H), 9.18-8.96 (br, 2H), 8.39(s, 1H), 8.33 (s, 1H), 7.98 (d, J=8.2 Hz, 1H), 7.95 (s, 1H), 7.91 (d,J=8.2 Hz, 1H), 7.75 (s, 1H), 7.60 (s, 1H), 7.57 (t, J=8.2 Hz, 1H), 7.46(t, J=8.2 Hz, 1H), 4.56-4.43 (br, 1H), 3.55-3.47 (br, 3H), 3.45 (s, 3H),3.43-3.34 (br, 2H), 3.13-2.99 (br, 2H), 2.27-2.09 (br, 4H). MS (ESI)calcd for [C₂₅H₂₇N₈O⁺]⁺: 455; found 455.

5,11-dimethyl-2-((1-(piperidin-4-yl)-1H-pyrazol-4-yl)amino)-5,11-dihydro-6H-benzo[e]pyrimido[5,4-b][1,4]diazepin-6-one

¹H NMR (400 MHz, DMSO-d₆) δ 9.77-9.60 (br, 1H), 9.16-8.94 (br, 2H), 8.35(s, 1H), 7.93 (s, 1H), 7.68 (dd, J=7.9, 1.8 Hz, 1H), 7.58 (s, 1H), 7.51(td, J=7.9, 1.8 Hz, 1H), 7.28 (d, J=7.9 Hz, 1H), 7.18 (t, J=7.9 Hz, 1H),4.52-4.44 (br, 1H), 3.42-3.34 (br, 5H), 3.38 (s, 3H), 3.10-3.00 (br,2H), 2.21-2.12 (br, 4H). MS (ESI) calcd for [C₂₁H₂₅N₈O⁺]⁺: 405; found405.

XMD18-83 was synthesized following similar strategies as shown in Scheme8.

4,5,11-trimethyl-2-((1-(piperidin-4-yl)-1H-pyrazol-4-yl)amino)-5,11-dihydro-6H-benzo[e]pyrimido[5,4-b][1,4]diazepin-6-one

¹H NMR (600 MHz, DMSO-d₆) δ 9.66-9.34 (br, 1H), 8.73-8.62 (br, 1H),8.53-8.38 (br, 1H), 7.89 (s, 1H), 7.63 (dd, J=7.8, 1.5 Hz, 1H), 7.56 (s,1H), 7.45 (td, J=7.8, 1.5 Hz, 1H), 7.20 (d, J=7.8 Hz, 1H), 7.14 (t,J=7.8 Hz, 1H), 4.45 (m, 1H), 3.42-3.28 (br, 5H), 3.18 (s, 3H), 3.06 (m,2H), 2.29 (s, 3H), 2.20-2.12 (br, 2H), 2.12-2.03 (br, 2H). MS (ESI)calcd for [C₂₂H₂₇N₈O]⁺: 419; found 419.

TABLE 1 Compounds of the invention Compound ID Structure SpectroscopyJWE-089

XMD16-34

XMD16-39

XMD16-43

XMD16-44

XMD16-45

XMD16-46

XMD16-58

XMD16-85

TABLE 2 Compounds of the invention EphA2 Activity (% Inhi- bition atCompound 10 micro- ID Structure molar) JWF-039

 358 JWF-040

 51.9 JWF-041

4850 JWF-042

 170 JWF-043

 113 JWF-044

 118 JWF-045

 162 JWF-046

  17.4 JWF-050

EphA2:  323 JWF-051

EphA2:   39.5 JWF-052

EphA2:  127 JWF-056

1810 JWF-057

   1.97 JWF-060

  24.0 XMD16-95

  21.6 XMD16-120

XMD16-101-1

XMD16-101-2

XMD16-117

XMD16-118

XMD16-121

XMD16-125

XMD16-122-1

XMD16-122-2

XMD16-127

  −1.1, −10.5 XMD16-128

  −5.1, −8.2 AB-1-9

AB-1-15

AB-1-16

AB-1-17

AB-1-24

AB-1-25

XMD16-117

XMD16-118

XMD16-123-1

XMD16-123-2

XMD16-124

TABLE 3 Compounds of the invention Compound ID Structure SpectroscopyXMD15-118

XMD15-119

XMD15-128

XMD15-129

XMD16-86

XMD16-35

XMD16-41

TABLE 4 Compounds of the invention Compound ID Structure SpectroscopyXMD15-143

XMD15-144

XMD15-145

XMD15-146

XMD16-91

TABLE 5 Compounds of the Invention Compound ID Structure SpectroscopyXMD16-12

XMD16-4

XMD17-109 (Compound 26)

XMD17-121

XMD17-133

XMD17-26 (Compound 25)

XMD17-27

XMD17-28

TABLE 6 Additional Compounds of the Invention Compound ID StructureSpectroscopy XMD11-85a

XMD11-85b

XMD11-85g

XMD11-85h

XMD11-85c

XMD11-85d

XMD11-85e

XMD11-85f

DLW-1-138-1

DLW-1-142-1

XMD-16-87

XMD-16-88

XMD-16-90

DLW-01- 080-01

XMD17-62

XMD17-63

XMD17-87

XMD8-81-1

XMD17-73

XMD18-19

XMD18-29

XMD18-30

XMD18-31

XMD18-33

XMD18-34

XMD18-36

XMD18-41

XMD18-42

XMD18-47

XMD18-48

XMD16-61

XMD16-62

XMD16-63

XMD16-64

XMD17-60

XMD17-61

HG-8-110-01

HG-8-112-01

HG-8-112-03

HG-8-126-01

HG-8-127-01

HG-8-137-01

HG-8-137-03

HG-8-138-01

HG-8-138-03

HG-9-43-01

DLW-01- 117-01

DLW-01- 124-01

JWE-035

JWE-036

JWE-037

JWE-038

JWE-041

JWE-042

JWE-043

JWE-044

JWE-045

JWE-067

JWE-068

JWE-071

JWE-094

XMD12-54

XMD16-10

XMD16-12

XMD16-116

XMD16-13

XMD16-144

XMD16-145

XMD16-146

XMD16-4

XMD16-47

XMD16-48

XMD16-5

XMD16-54

XMD16-55

XMD16-56

XMD16-67

XMD16-68

XMD17-1

XMD17-109

XMD17-121

XMD17-122

XMD17-123

XMD17-124

XMD17-133

XMD17-134

XMD17-137

XMD17-139

XMD17-140

XMD17-141

XMD17-16

XMD17-26

XMD17-27

XMD17-28

XMD17-35

XMD17-37

XMD17-38

XMD17-51

XMD17-78

XMD17-81

XMD17-85

XMD17-86

XMD17-88

XMD17-89

DLW-01- 122-01

DLW-01- 125-01

XMD17-75

XMD17-77

XMD17-79

DLW-01- 126-01

XMD17-82

DLW-01- 111-01

DLW-1- 141-1

DLW-1- 138-1

DLW-1- 142-1

XMD11-138

XMD11-139

XMD11-140

XMD11-141

XMD12-10

XMD12-129

XMD12-130

XMD12-2

XMD12-3-1

XMD12-3-2

XMD12-43a

XMD12-51

XMD12-52

XMD12-53

XMD12-54

XMD12-55

XMD12-68

XMD12-69

XMD12-70-2

XMD13-137

XMD13-37

XMD13-42

XMD13-43

XMD13-44

XMD13-65

XMD13-66

XMD13-93

XMD13-98

XMD15-69

XMD16-11

XMD16-91

HG-9-75- 06

HG-9-135- 01

HG-9-129- 01a

HG-10-66- 01

HG-10-67- 01

HG-10-67- 02

HG-10-67- 03

HG-10-68- 01

HG-10-69- 01

HG-10-75- 01

HG-10-75- 02

XMD7-126

XMD7-126

XMD7-127

XMD8-75

XMD8-76

XMD8-81

XMD8-87

XMD8-91

XMD8-96

XMD8-97

XMD8-98

XMD9-18

XMD9-21

XMD10-124

XMD10-127

XMD10-129

XMD11-48

XMD11-55

XMD11-56

XMD11-58

XMD17-87

XMD17-67

HG9-27-02

HG8-140-02

HG8-111-01

HG8-111-02

HG9-29-01

HG9-27-02

HG9-29-02

HG9-29-03

HG9-29-04

HG9-29-05

HG9-48-01

HG9-95-01

HG9-95-02

HG9123-01

HG9123-02

HG9123-03

HG9123-04

¹H NMR (600 MHz, CD₃OD) δ 8.07 (s, 1H), 7.70 (dd, J = 1.8, 7.8 Hz, 1H),7.65 (s, 1H), 7.46 (dt, J = 1.8, 7.8 Hz, 1H), 7.16 (d, J = 8.4 Hz, 1H),7.13 (t, J = 7.8 Hz, 1H), 6.86 (s, 1H), 3.61 (t, J = 7.2 Hz, 2H), 3.41(s, 3H), 3.31 (s, 3H), 2.88 (t, J = 7.2 Hz, 2H). MS (ESI) m/z 350 (M +H)⁺.

¹H NMR (600 MHz, CD₃OD) δ 8.14 (s, 1H), 7.78 (d, J = 7.8 Hz, 1H), 7.56(t, J = 7.8 Hz, 1H), 7.28-7.24 (m, 2H), 4.16 (brs, 1H), 3.62 (d, J =12.0 Hz, 2H), 3.47 (s, 3H), 3.44 (s, 3H), 3.20 (t, J = 12.6 Hz, 2H),2.91 (s, 3H), 2.34 (d, J = 13.2 Hz, 2H), 1.87 (q, J = 13.2 Hz, 2H). MS(ESI) m/z 353 (M + H)⁺.

¹H NMR (600 MHz, CD₃OD) δ 8.17 (s, 1H), 8.12 (d, J = 9.0 Hz, 1H), 7.71(dd, J = 1.2, 7.8 Hz, 1H), 7.45 (dt, J = 1.8, 7.8 Hz, 1H), 7.17 (d, J =8.4 Hz, 1H), 7.13 (t, J = 7.8 Hz, 1H), 6.67 (d, J = 2.4 Hz, 1H), 6.60(dd, J = 3.0, 9.0 Hz, 1H), 4.64- 4.60 (m, 1H), 3.74-3.70 (m, 1H),3.48-3.44 (m, 2H), 3.43 (s, 3H), 3.36 (s, 3H), 2.84-2.80 (m, 2H), 1.97-1.95 (m, 2H), 1.69-1.63 (m, 2H), 1.33 (d, J = 6.6 Hz, 6H), MS (ESI) m/z489 (M + H)⁺.

1H NMR (600 MHz, CD3OD) δ 8.11 (d, J = 8.4 Hz, 1H), 7.99 (s, 1H), 7.88(d, J = 7.2 Hz, 1H), 7.44 (dd, J = 1.2, 7.8 Hz, 1H), 7.16 (d, J = 1.2Hz, 1H), 7.14-7.11 (m, 2H), 4.70 (brs, 1H), 4.39 (t, J = 8.4 Hz, 2H),3.96 (s, 3H), 3.55-3.45 (m, 4H), 3.41 (s, 3H), 3.40- 3.32 (m, 6H),3.25-3.21 (m, 4H), 2.65 (s, 3H), 2.20-1.95 (m, 2H), 1.75- 1.65 (m, 2H).MS (ESI) m/z 583 (M + H)+.

1H NMR (600 MHz, CD3OD) δ 8.49 (d, J = 8.4 Hz, 1H), 8.36 (s, 1H), 7.64(dd, J = 1.8, 7.8 Hz, 1H), 7.48-7.45 (m, 1H), 7.27 (d, J = 8.4 Hz, 1H),7.18 (t, J = 7.8 Hz, 1H), 7.08-7.07 (m, 2H), 4.82-4.78 (m, 1H), 4.19 (q,J = 7.2 Hz, 2H), 3.55-3.48 (m, 9H), 3.45- 3.30 (m, 6H), 3.29-3.25 (m,2H), 2.93 (s, 3H), 2.36-2.33 (m, 1H), 2.15- 2.10 (m, 3H), 1.70-1.57 (m,6H), 1.56-1.54 (m, 1H), 1.47 (t, J = 7.2 Hz, 3H). MS (ESI) m/z 639 (M +H)+. XMD18-83

HTH-01-015

Example 9: Mps1 (TTK) Cellular Assay—Mitotic Escape Assay

Hela (or U2OS cells) were plated at roughly 30-35% cell density. After24 hours the medium* was removed and fresh medium supplemented with 2.5mM thymidine was added to arrest cells at the G1/S transition. After 24hours in thymidine block the medium was removed, the cells were washed3× with PBS and replaced with medium supplemented with 330 nM nocodazole(Noc). The cells were incubated with nocodazole for 16-18 hours toproduce a mitotic arrest. The medium was then removed carefully andreplaced with medium supplemented with 330 nM nocodazole and testcompound at the desired concentration (with the final concentration ofDMSO below 0.2%). After 2 hours, the cells were harvested, lysed in RIPAbuffer, and the levels of cyclin B or phosphorylated Histone 3 (Ser10)determined by western blotting. Alternatively, cells were treated oncoverslips, fixed, and phosphorylated Histone 3 levels determined byimmunofluorescence. *Hela/U2OS medium—Dulbecco's Modified Eagle's Medium(DMEM, Sigma), 10% fetal bovine serum, 1% penicillin/streptomycin

Example 10: Plk1 Cellular Assay—Mitotic Arrest Assay

Hela cells were plated at roughly 80% cell density on poly-lysine coatedglass coverslips. After 24 hours the medium* was removed and freshmedium supplemented with test compounds was added. Twenty-four hourspost-treatment the medium was removed, the coverslips were washed oncewith phosphate-buffered saline (PBS), pH 7.4 and the cells fixed for 10minutes at room temperature using the following fixative solution: 100mM K-Pipes, pH 6.8, 10 mM EGTA, 1 mM MgCl₂, 0.2% Triton X-100, 3%formaldehyde. The coverslips were washed 3× with Tris-buffered salinesolution (50 mM Tris-HCl pH 7.4, 150 mM NaCl) containing 0.1% TritonX-100 (TBST). The samples were blocked using 2% bovine serum albumin(BSA) in TBST. The samples were then incubated with aphosphorylation-specific antibody against histone 3 (phospho H3)serine-10 (Upstate, 1:500-1:1000) in blocking solution. Cells can alsooptionally be stained for tubulin as well using appropriate antibodies.After a 2-hr. incubation at room temperature (or 4° C. overnight), thesamples were washed 3× with TBST. The samples were then incubated withan appropriate secondary antibody in blocking solution for 1-2 hrs atroom temperature (or 4° C. overnight). The samples were washed 3× withTBST and then incubated with Hoechst 33342 stain (Invitrogen,1:1000-1:2000) in TBST for 15 minutes at room temperature. The sampleswere washed 3× with TBST and mounted onto glass slides using ProlongGold Antifade Reagent (Invitrogen). *Hela/U2OS medium—Dulbecco'sModified Eagle's Medium (DMEM, Sigma), 10% fetal bovine serum, 1%penicillin/streptomycin

Example 11: In Vitro Mps1 Kinase Assay—Invitrogen Mps1 (TTK)LanthaScreen Activity Assay

Kinase reactions were carried out at room temperature with the followingcomponents: 1× kinase reaction buffer, 5 μg/mL (40 nM) Mps1 kinase, 200nM AF-647 E4Y substrate, and 1 μM ATP (K_(m,app)<1 μM). After one hour apreparation of EDTA (20 mM) and Eu-PY20 Tb-labeled antibody (4 nM) inTR-FRET dilution buffer was added. The final concentration of EDTA andEu-PY20 in the reaction mixture is 10 mM and 2 nM respectively. Thereaction mixture was incubated at room temperature for 30 minutes beforebeing read on a plate reader configured for LanthaScreen™ TR-FRET.Kinase reactions were run over several concentrations of inhibitor toobtain dose-dependent curves.

Example 12: Kinase Selectivity Analysis

The SAR exploration of the benzo[e]pyrimido-[5,4-b]diazepine-6(11H)-onescaffold led to the discovery of the relatively LRRK2 selectiveinhibitor 24 and ERK5 selective inhibitor 26 (FIG. 1). The structuralfeatures of N-methyl substitution at lactam position (R²), the 2-ethoxygroup of 4-amide substituted aniline, N-cyclopentyl substitution (X) andno substituent (R⁴═H) on the aryl ring of anthranilic acid wereessential to achieve potent cellular inhibitory activity against ERK5and high specificity (FIG. 1, highlighted in red). The linkage (R⁶) ofindoline-7-carboxylic group exhibited improved LRRK2 selectivityexemplifying by compound 24 (FIG. 1, highlighted in blue). Theintroduction of amide functional group at the 4-position of 2-anilinomoiety is favorable for both ERK5 and LRRK2. The substituent at theortho-position of 2-anilino moiety (R⁵) and the linkage group (X) arekey structural features to separate the SAR of thebenzo[e]pyrimido-[5,4-b]diazepine-6(11H)-ones for ERK5 and LRRK2 (FIG.3A and FIG. 3B). Certain pyrimidine derivatives,(4-((5-chloro-4-(methylamino)pyrimidin-2-yl)amino)-3-methoxyphenyl)(morpholino)methanone(HG-10-102-01) and 2-arylmethyloxy-5-subtitutent-N-arylbenzamide(GSK2578215A) have been reported that are potent LRRK2 inhibitors andthat do not inhibit ERK5.

Cellular LRRK2 Inhibitory Effect of Compound 24 and 26.

We examined the abilities of compounds 24 and 26 to inhibit LRRK2 in acellular context. As there are no validated direct phosphorylationsubstrates of LRRK2, we monitored phosphorylation of Ser910 and Ser935,two residues whose phosphorylation is known to be dependent upon LRRK2kinase activity (FIG. 2A and FIG. 2B). Compound 24 induced adose-dependent inhibition of Ser910 and Ser935 phosphorylation in bothwild-type LRRK2 and LRRK2[G2019S] stably transfected HEK293 cells (FIG.2A). Significant reduction on the level of phosphrylation of residesSer910 and Ser935 was observed at 1-3 μM of 24 for wild-type LRRK2 andat slightly lower doses for LRRK2[G2019S] (FIG. 2A), which isapproximately the same potency relative to LRRK2-IN-1. Compound 24 hadno effect on the phosphorylation of Ser910 and Ser935 at a concentrationof up to 3 μM in the drug-resistant LRRK2[G2019S+A20161] andLRRK2[A20161] mutants (FIG. 2A), revealing that 24 has the same activityprofile compared to LRRK2-IN-1. Consistent with the biochemical results,compound 26 didn't show any inhibitory effect against LRRK2 at aconcentration of up to 3 μM in this cellular context (FIG. 2B).

We next examined the effects of compounds 24 and 26 on endogenouslyexpressed LRRK2 in human lymphoblastoid cells derived from a control andParkinson's disease patient homozygous for the LRRK2[G2019S] mutation(FIG. 3A-FIG. 3B). We found that increasing doses of 24 led to similarreduction on the levels of phosphorylation of endogenous LRKK2 at Ser910and Ser935, as was observed in HEK293 cells stably expressing wild-typeLRRK2 or LRRK2[G2019S] (compare FIG. 2A to FIG. 3A). Moreover, 24 wasalso more potent against LRRK2[G2019S] mutant than wild type LRRK2,which is consistent with the trend we observed in HEK293 cells.Similarly, compound 26 didn't show inhibitory effects on endogenousLRRK2 (compare FIG. 2B to FIG. 3B). Taken together, compound 24 is aspotent LRRK2 inhibitor as LRRK2-IN-1 and worked both in vitro and incells and with improved selectivity towards LRRK2. Compound 26 is a ERK5specific inhibitor, which has at least 30-fold cellular selectivity forERK5 relative to LRRK2 and should not inhibit LRRK2 when used at 1 μMconcentrations.

We assessed the selectivity of this scaffold using the KINOMEscanmethodology across a near comprehensive panel of 442 kinases. Compounds24, 25 and 26 were screened at a concentration of 10 μM which revealed ahighly selective profile for this inhibitor class. The structure ofCompound 24 is shown below:

Compound 26 having ortho-ethoxy aniline demonstrated outstandingselectivity with a KINOMEscan selectivity score of S₁₀ of 0.007 (3/442),and only interactions with ERK5, doublecortin and CaM kinase-like 2(DCAMKL2) and polo-like kinase 4 (PLK4) were detected. Compound 25having ortho-methoxy aniline exhibited a S₁₀ of 0.018 (8/442). Theseresults revealed that the ortho-substituent could serve as theselectivity handle. Compared with our previously reported ERK5inhibitor, XMD8-92 (11, S₁₀=0.012, 5/402), compound 26 represents afurther improvement in selectivity. Compound 24 exhibited the sameKINOMEscan selectivity score of S₁₀ of 0.036 (16/442) as that of ourprevious LRRK2 inhibitor (LRRK2-IN-1), while being more selective forLRRK2 over ERK5. Compounds 25 and 26 were also profiled against selectedpanels of kinases in HeLa and PC3 cell lysates using a chemicalproteomics approach, KiNativ. These results revealed that only ERK5(ERK5) was inhibited with higher than 90% activity at a concentration of10 μM for both 25 and 26, which further confirmed their highly selectiveprofiles.

To better understand the SAR for LRRK2, we performed a molecularmodeling study using Glide based upon the recently reported crystalstructure of Roco kinase (PDB accession code: 4F1T) (FIG. 4A, FIG. 4B,and FIG. 4C). This model allows explanation of some of the SAR that weobserved. Overall 26 is predicted to bind to LRRK2 in a manner analogousto what has been observed for a structural analogue, Mps1-IN-2, bound toTTK: The tricyclic core of the compound curves around Leu2001 in thebase of the ATP binding site, forming two hydrogen bonds with the hingeregion at Ala1950, while the piperidin-piperazine goes towards thesolvent region (FIG. 4A). The cyclopentyl group points towards theglycine rich loop, against Leu1885, and would appear to force thetricyclic ring towards the base of the ATP binding site.

The SAR suggests that the phenyl ring of the 2-amino moiety hasimportant interactions, as alternative substituents lost activity toLRRK2 and ERK5, and in the model this moiety would bind against thehinge region. The decrease in LRRK2 affinity caused by N-substitutionwith increasing size up to cyclopentyl may be because the tricyclingring is forced into a less favourable contact with Ala2016 as theN-substituent makes contact with the glycine-rich loop at Leu1885. Thesame contacts with Ala2016 and nearby residues would explain whysubstitution of the anthranilic acid may result in weaker binding.Ortho-substitution of the aniline with increasingly large groups (ethyl,isopropyl) resulted in decreased affinity for LRRK2. Since this groupwould bind adjacent to Leu1949 (FIG. 4B, FIG. 4C) only conformationswith the additional carbons pointing away from Leu1949 would befavourable, resulting in increasingly unfavourable entropy as thesubstituent goes from methoxy to ethoxy to isopropoxyl.

The key active site residues in this discussion above are eitherconserved in ERK5 (Leu1949, Leu2001) or conservatively substituted(Leu1885, Ala2016, Met1947). We have recently determined a crystalstructure of ERK5 bound to 25 which confirms the both the binding modelfor 26 with LRRK2 and the SAR explanation.

Conclusions

The new chemo-type of benzo[e]pyrimido-[5,4-b]diaze pine-6(11H)-onerepresents a privileged scaffold for developing ERK5 and LRRK2 kinaseinhibitors. Comprehensive SAR exploration led to the identification ofthe key structural features to separate the SAR of this scaffold forERK5 and LRRK2. Compound 24 is as potent a LRRK2 inhibitor asLRRK2-IN-1, and worked both in vitro and in cells and with improvedselectivity towards LRRK2. Compound 26 represents the most selective andpotent ERK5 inhibitor we have developed so far. Given the outstandingspecificity and excellent cellular efficacy, 26 could serve as aversatile tool to further probe ERK5 biology. Thebenzo[e]pyrimido-[5,4-b]diaze pine-6(11H)-ones with excellentselectivity, favorable pharmacokinetic parameters, and great efficacy inxenograft tumor models can serve as a privileged template to developtherapeutic agents targeting ERK5.

ERK5 Autophosphorylation Assay.

HeLa cells were serum starved overnight followed by treatment withinhibitors for one hour. Cells were then stimulated with EGF (20 ng/mL)for 17 min and harvested in RIPA buffer (1×PBS, 1% NP40, 0.5% sodiumdeoxycholate, 0.1% SDS, 0.1 mg/ml PMSF and 1 mM sodium orthovanadate).Proteins from total cell lysates were resolved by 6% sodium dodecylsulfate (SDS)-poly-acrylamide gel electrophoresis (PAGE), transferred tonitrocellulose membrane, blocked in 5% nonfat milk, and blotted withanti-ERK5 antibody.

Baculovirus Expression of Active ERK5 and Purification.

pFastBAC vector encoding N-terminal hexahistidine-tagged human ERK5 andHA-tagged human MEK5-DD (constitutively active) were used to generaterecombinant baculovirus using the Bac-to-Bac system (Invitrogen).Spodoptera frugiperda 21 cells (1.5×10⁶/ml) were infected at amultiplicity of infection of 6 with a mix of both baculovirus andharvested 72 h post-infection. Pelleted cells were lysed in ice-coldlysis buffer (50 mM Tris/HCl, pH 7.5, 1 mM EGTA, 1 mM EDTA, 1 mM sodiumorthovanadate, 10 mM sodium β-glycerophosphate, 50 mM NaF, 5 mM sodiumpyrophosphate, 0.27 M sucrose, 1 mM benzamidine, 2 mMphenylmethanesulphonylfluoride (PMSF) and 1% Triton X-100), lysed in oneround of freeze/thawing, sonicated (4×20 s) and centrifuged at 25,000 gfro 30 min. His-tagged ERK5 was purified as described for His-taggedBRSK1,³⁷ using 5 ml Ni-NTA-agarose resin (Qiagen) followed by gelfiltration chromatography on Superdex 200HR column on an AKTA system (GEHealthcare). Active ERK5 was purified with yields of ˜5 mg/L of infectedcells, and was greater than 90% homogeneous as judged by densitometricscanning of Coomassie Blue-stained SDS/PAGE gels.

ERK5 Kinase Activity In Vitro Assay.

Kinase activity was determined in an assay volume of 40 μl in kinasebuffer (50 mM Tris-HCl, pH 7.5, 0.1 mM EGTA, 1 mM 2-mercaptoethanol)containing 200 ng of pure active ERK5 and the indicated amount ofinhibitor. Reaction started by adding 10 mM magnesium acetate, and 50[γ-³²P]-ATP (500 cpm/pmol) and 250 μM PIMtide (ARKKRRHPSGPPTA) assubstrates. Assays were carried out for 20 min at 30° C., terminated byapplying the reaction mixture onto p81 paper and the incorporatedradioactivity measured as described previously.

Adaptor Kinase Assay of LRRK2 [G2019S].

In vitro kinase assays were conducted at Invitrogen (Madison, Wis.)using the SelectScreen Kinase Profiling Service.

LRRK2 Cellular Assay.

Reagents and General methods. Tissue-culture reagents were from LifeTechnologies. Protein G Sepharose was from Amersham. DNA constructs usedfor transfection were purified from Escherichia coli DH5α using Qiagenor Invitrogen plasmid Maxi kits according to the manufacturer'sprotocol. All DNA constructs were verified by DNA sequencing, which wasperformed by The Sequencing Service, School of Life Sciences, Universityof Dundee, Scotland, U.K., using DYEnamic ET terminator chemistry(Amersham Biosciences) on Applied Biosystems automated DNA sequencers.

Cell culture, treatments and cell lysis. HEK293 was cultured in DMEM(Dulbecco's Modified Eagle's medium) supplemented with 10% FBS (fetalbovine serum), 2 mM glutamine and 1× penicillin/streptomycin solution.Lymphoblastoid cell lines were generated by EBV (Epstein-Barr virus)transformation of B lymphocytes using standard methods (EuropeanCollection of Cell Cultures). Cell-line ANK is derived from a47-year-old individual homozygous for the LRRK2[G2019S] mutation whopresented with Parkinson's disease. Cell-line AHE is derived from a31-year-old individual, lacking mutation at the LRRK2 Gly²⁰¹⁹ residue,and presented with no disease. Human lymphoblastoid cells weremaintained in RPMI 1640 with 10% FBS, 2 mM glutamine, 1×penicillin/streptomycin solution and were maintained at cell density of0.3×10⁶-2×10⁶ cells per ml. Epstein-Barr virus immortalized primaryhuman lymphoblastoid cells from one control subject and one Parkinson'sdisease patient homozygous for the LRRK2 [G2019S] mutation were kindlyprovided by Alastair Reith (GSK) and have been described previously. Forinhibitor experiments, compounds were dissolved in DMSO and utilized atthe indicated concentrations. The concentration of DMSO in the culturemedia did not exceed 1%. Following treatment, cells were washed oncewith phosphate buffered saline (PBS) buffer and lysed with lysis buffer(50 mM Tris/HCl, pH 7.5, 1 mM EGTA, 1 mM EDTA, 1 mM sodiumorthovanadate, 10 mM sodium β-glycerophosphate, 50 mM NaF, 5 mM sodiumpyrophosphate, 0.27 M sucrose, 1 mM benzamidine, 2 mMphenylmethanesulphonylfluoride (PMSF) and 1% Triton X-100). When notused immediately, all lysate supernatants were snap-frozen in liquidnitrogen and stored at −80° C. until use. Protein concentrations weredetermined following centrifugation of the lysate at 16,000×g at 4° C.for 20 minutes using the Bradford method with BSA as the standard.Transient transfection of HEK 293 cells was performed using thepolyethyleneimine (PEI) method.

Immunoblot Procedures.

Cell lysates from human lymphoblastoid cells and GFP-LRRK2 expressingstable cell lines were eluted in 65 μl 2×LDS sample buffer (Invitrogen)with final concentration of 1 μg/μl. Following heating at 70° C. for 10min, 15 μl aliquots were resolved on 8% SDS polyacrylamide gels andtransferred to nitrocellulose membranes for detection of LRRK2phosphorylated at Ser910, LRRK2 phosphorylated at Ser935 and totalLRRK2, using purified rabbit monoclonal antibodies (LRRK2 phospho-serine910 clone, LRRK2 phospho-serine 935 clone and LRRK2 100-500 clone) inPBS with 0.1% sodium azide (Epitomics). Immunoblot films were scanned onan Epson 4990 scanner, and images were managed with Adobe Photoshop.

Molecular Docking Study

A molecular docking study to elucidate the interaction between theinhibitors with the LRRK2 kinase domain was performed. First, weconstructed the homology model structure of the LRRK2 kinase domain. Weused a crystal structure of Roco kinase (PDB accession code: 4F1T).Sequence alignment of LRRK2 and template proteins was generated usingthe Discovery Studio 3.5 package (http://www.accelrys.com). A 3D modelstructure of LRRK2 was built by using the Modeller in Discovery Studio3.5 package and was further refined by using the CHARMM force field.Second, compounds 25 and 26 were built using Maestro build panel andminimized using the Impact module of Maestro in the Schrödinger suiteprogram. The LRRK2 structure was minimized using the Protein PreparationWizard by applying an OPLS force field. For the grid generation, thebinding site was defined as the centroid of the ATP binding site. Liganddocking into the active site of LRRK2 was carried out using theSchrödinger docking program, Glide. The best-docked poses were selectedas the lowest Glide score. The molecular graphics for the inhibitorbinding pocket and refined docking models were generated using PyMolpackage (http://www.pymol.org).

Example 13: In Vitro EphA2 Kinase Assay—Invitrogen EphA2 Z′-LYTEActivity Assay

EphA2 kinase assays were performed according to the methods described inZ′-LYTE® Screening Protocol and Assay Conditions at the LifeTechnologies website (available at http://www.invitrogen.com).

Western Analysis XMD16-95 Inhibition of EphA2 Kinase Activity In Vivo:

A375 cells were treated with EphrinA2 (100 ng/mL) (R&D systems, Cat No.7856-A2-050) for 15 minutes, and test compounds were added to cellculture for 60 minutes treatment. Standard western analysis wasperformed to monitor the EphA2 phosphorylation (Y594) (antibody fromCell Signaling Technology, Cat. No. 3970) status.

The activity of certain compounds of Formula II against EphA2 and otherkinases is shown in Table 7.

TABLE 7 ActivX XMD16-123-1 EphA1 EphA2 EphA2 EphA7 EphB2 EphB4 8.5 9.310.5  15.3  2.5 6.8

XMD16-125 EphA1 EphA2 EphA2 EphA7 EphB2 EphB4 −19.4  0.9 1.6 −7.9  0.9−14.6 

XMD16-121 EphA1 EphA2 EphA2 EphA7 EphB2 EphB4 −2.6  5.3 −4.4  2.3 12.2 15.9 

XMD16-127 EphA1 EphA2 EphA2 EphA7 EphB2 EphB4 1.9 −10.5  −1.1  1.7 2  6.6

XMD16-128 EphA1 EphA2 EphA2 EphA7 EphB2 EphB4 −2.1  −5.1  −8.2  −15.4 4.5 −13.7 

XMD16-118 EphA1 EphA2 EphA2 EphA7 EphB2 EphB4 −12.9  3.9 −8.3  −17.1 −20.9  −22.3 

XMD16-120 EphA1 EphA2 EphA2 EphA7 EphB2 EphB4 49.2 41.1 44.1 14.7 60.172.5

AB-1-9 EphA1 EphA2 EphA2 EphA7 EphB2 EphB4 35.6  10.5  41.7  −35.6 −10.8  10  

AB-1-15 EphA1 EphA2 EphA2 EphA7 EphB2 EphB4 1.2 −8.8  −5   2.3 5.2 −2.4 

AB-1-16 EphA1 EphA2 EphA2 EphA7 EphB2 EphB4 18   −6.8  −1.8  2.8 7  10.7 

XMD16-101-1 EphA1 EphA2 EphA2 EphA7 EphB2 EphB4 22.3  16   8.2 −1.4  6.13.9

XMD16-101-2 EphA1 EphA2 EphA2 EphA7 EphB2 EphB4 9.1 8.1 1.9 −11.4  0.6−1.1 

AB-1-17 EphA1 EphA2 EphA2 EphA7 EphB2 EphB4 56.8  33   30.8  3.4 21.5 39.8 

XMD16-95 EphA1 EphA2 EphA2 EphA7 EphB2 EphB4 89.1  91.7  76.6  80.7 97.2  95.7 

XMD16-122-1 EphA1 EphA2 EphA2 EphA7 EphB2 EphB4 −12.8  6.2 −2.7  −8.3 −13    −4.3 

XMD16-122-2 EphA1 EphA2 EphA2 EphA7 EphB2 EphB4 −4.5  −2.5  −11.3  0.5−5.5  2  

XMD16-124 EphA1 EphA2 EphA2 EphA7 EphB2 EphB4 −4.7  5.8 −18.4  4.3 18.3 −17.3 

AB-1-24 EphA1 EphA2 EphA2 EphA7 EphB2 EphB4 28.6  5.2 4.9 12.1  6.8 7.4

XMD16-117 EphA1 EphA2 EphA2 EphA7 EphB2 EphB4 13.6  6.8 9   5   19.8 6.2

Ambit XMD16-117 EPHA1 EPHA2 EPHA3 EPHA4 EPHA5 EPHA6 EPHA7 EPHA8 EPHB1EPHB2 EPHB3 EPHB4 EPHB6 69   100    47   84   91   92   100    100   91   100    93   91   20  

XMD16-124 EPHA1 EPHA2 EPHA3 EPHA4 EPHA5 EPHA6 EPHA7 EPHA8 EPHB1 EPHB2EPHB3 EPHB4 EPHB6 81   93   77   89   90   100    100    100    84  100    93   99   78  

INCORPORATION BY REFERENCE

The contents of all references (including literature references, issuedpatents, published patent applications, and co-pending patentapplications) cited throughout this application are hereby expresslyincorporated herein in their entireties by reference. Unless otherwisedefined, all technical and scientific terms used herein are accorded themeaning commonly known to one with ordinary skill in the art.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents of the specificembodiments of the invention described herein. Such equivalents areintended with be encompassed by the following claims.

What is claimed:
 1. A compound having the formula:

or a pharmaceutically acceptable salt thereof, wherein, R′ is H ormethyl; L is absent, SO₂, or CO; X is H, methyl, fluoro, chloro, phenyl,pyridinyl, thiophenyl, —NH—CO—CH═CH—CH₂—N(CH₃)₂, —S(O)₂—N(CH₃)₂, or

Z is NH or N—C1-C3 alkyl; R₂ is hydrogen or methyl; R₆ is hydrogen; andR₁ is H, C═O(CH₃),


2. The compound of claim 1, wherein the compound is selected from thegroup consisting of:


3. A compound selected from the group consisting of:


4. The compound

or a pharmaceutically acceptable salt thereof.
 5. The compound

or a pharmaceutically acceptable salt thereof.